Sixteen Democrats said Monday that they will oppose Rep. Nancy Pelosi’s bid for House speaker, an act of defiance that puts the dissidents on the cusp of forcing a seismic leadership shake-up as the party prepares to take the majority.

Their pledge to oppose Pelosi, delivered in a letter to Democratic colleagues, comes as the California congresswoman has marshaled a legion of supporters to make her case. She is courting members in one-on-one conversations while securing the backing of allies on and off Capitol Hill.

On Monday, the International Association of Fire Fighters; UnidosUS, a leading Latino civil rights organization; and nine military veterans serving in the House endorsed her return as speaker. Meanwhile, at a town hall meeting in Massachusetts, a leader of her opposition in the House was confronted by dozens of pro-Pelosi protesters.

The insurrection against Pelosi stands as the only official obstacle to unity in the top leadership ranks, after Rep. Diana DeGette (D-Colo.) on Monday withdrew her challenge for the No. 3 job held by Rep. James E. Clyburn (D-S.C.), and Pelosi endorsed her leadership team. But the renegades’ threat is considerable.

“We are thankful to Leader Pelosi for her years of service to our Country and to our Caucus,” her opponents wrote. “However, we also recognize that in this recent election, Democrats ran on and won on a message of change.”

Pelosi, 78, has expressed complete confidence that she will retake the speaker’s gavel in January — eight years after she lost it following massive Republican gains in the 2010 midterms and 16 years after she was first elevated to the top Democratic leadership post in the House.

Pelosi is the only speaker candidate who has declared ahead of a party nominating vote on Nov. 28, with a vote of the full House on Jan. 3. But her opponents believe that an alternative will emerge if it becomes clear Pelosi does not have the 218 votes needed to win in January.

Drew Hammill, a Pelosi spokesman, said Monday that she “remains confident in her support among members and members-elect” and noted that more than 90 percent of the incoming Democratic caucus declined to sign the letter.

Two Democratic aides said Pelosi inquired with at least one incoming member about whether they would be willing to vote present — a move that would lower the threshold for becoming speaker while also allowing Pelosi’s critics to keep their word in opposing her. Hammill declined to comment on whether Pelosi is exploring that possibility.

The signers of the letter might not be able to force Pelosi out themselves, but there is at least a handful of Democrats who plan to oppose her but did not sign the letter.

The size of the Democratic majority remains in flux, but Democrats have won 232 seats, according to the Associated Press, with four races still undecided. If the current leads hold in those uncalled races, Democrats will have won 234 seats — a 17-seat majority.

Analysis | Does Pelosi have the votes to become Speaker?

That means Pelosi could lose as many as 16 Democratic votes when she stands for election as speaker Jan. 3. Two of the 16 signers, Anthony Brindisi (New York) and Ben McAdams (Utah), have yet to be declared winners.

Also signing the letter were Reps. Jim Cooper (Tenn.), Bill Foster (Ill.), Seth Moulton (Mass.), Brian Higgins (N.Y.), Stephen F. Lynch (Mass.), Ed Perlmutter (Colo.), Kathleen Rice (N.Y.), Tim Ryan (Ohio), Linda T. Sánchez (Calif.), Kurt Schrader (Ore.), Filemon Vela (Tex.), and newly elected members Joe Cunningham (S.C.), Max Rose (N.Y.) and Jeff Van Drew (N.J.).

Not signing the letter was Rep. Marcia L. Fudge (D-Ohio), who has publicly opposed Pelosi and is mulling a run against her.

A spokesman for Fudge said Monday that she was unavailable for an interview and spending time in her congressional district in the Cleveland area, adding that her final decision on the speaker race is not expected until after the Thanksgiving holiday.

A Fudge associate, who spoke on the condition of anonymity to discuss private conversations, said it was unclear why Fudge did not sign the letter but that the lawmaker was deliberately keeping a low profile this week as she continues to think through her possible candidacy, with the travel and fundraising of the job weighing on her and potentially pushing her away from running.

Moulton, one of the leaders of the insurrection, stayed in close touch with fellow Pelosi critics on Monday, including a call with Fudge, who has been his mentor in the House, according to a person familiar with the conversation. Moulton continues to encourage her to run, the person said.

At a town hall meeting in Amesbury, Mass., Monday night, Moulton heard an earful from Pelosi supporters — including about 20 organized protesters who held signs reading “I Stand With Nancy.” Many of them saw sexism and ageism in the push to oust Pelosi.

“I almost feel like I’m targeted — I’m old and I’m a woman,” said Lynda Christian, 80, of nearby Burlington, Mass. “Nancy Pelosi, I don’t know her, but she’s done a fantastic job. Who fires somebody who does something well?”

Addressing the sometimes testy crowd, Moulton said it would be “failing the American people” to keep Pelosi in place after 16 years in charge.

“The American people cried out for change in this election,” he added. “What’s going on in Washington isn’t working, and we need a new generation of leadership.”

Five other Democrats — Rep. Conor Lamb (Pa.) and Reps.-elect Jason Crow (Colo.), Jared Golden (Maine), Mikie Sherrill (N.J.) and Abigail Spanberger (Va.) — have made firm statements saying they would not vote for Pelosi but did not sign the letter.

Representatives for Crow, Lamb and Spanberger each said Monday that their positions on Pelosi had not changed and that they intend to vote against her in the January floor vote.

Golden said the same in an interview, saying, “It’s your vote, not signing a piece of paper, that counts.”

“We need to pass the torch to new leaders in the Democatic Party, and I stand by that, and I’m going to vote that way in caucus and on the House floor,” he said.

In her conversations with individual members, Pelosi has started inquiring about their policy priorities and committee assignment preferences. The Democratic Caucus gives its top leader wide leeway to make those decisions, crucial to a lawmaker’s legislative career.

She has also met with key subgroups within the caucus, including hearing out the left-leaning Congressional Progressive Caucus and the more centrist New Democrat Coalition. Both groups expect to have scores of Democrats in their ranks next year.

The Progressive Caucus issued a statement last week supportive of Pelosi, citing her pledge to help appoint caucus members to coveted committees such as Appropriations, Financial Services, and Ways and Means.

The New Democrats are pressing Pelosi for rules changes to ensure lawmakers have time to read bills and that operations in the House are more transparent, and Pelosi attended a meeting of the group Friday during which she was pressed on the need to shield centrist members from politically perilous votes.

Rep. Jim Himes (D-Conn.), the chairman of the coalition, cited the cap-and-trade climate bill that House Democrats passed in 2009, only to see it languish in the Senate. Many Republican challengers used the bill in their campaign to oust Democratic incumbents the next year.

“The culture of our caucus is very progressive, as it should be. But we need to make sure that we don’t do things which damage the electoral prospects of the people who gave us the majority,” he said. “I think she heard us loud and clear.”

DeGette had challenged Clyburn, the highest-ranking African American in House leadership, for the majority whip post. But facing a backlash from members of the Congressional Black Caucus, DeGette dropped her bid — ensuring, for now, that Clyburn will remain in the top ranks.

Any major shake-up in the Democratic leadership now depends on whether the dissidents can muster the votes to keep Pelosi from seizing the House speaker’s gavel in January. If that bid is successful, it could kick off a wholesale scramble that could also threaten Clyburn, the current assistant Democratic leader, and Steny H. Hoyer (D-Md.), who is seeking to move from whip to majority leader.

DeGette, who has long served as chief deputy whip, launched her campaign after the Nov. 6 election by touting her experience in rounding up support for difficult pieces of legislation — implicitly making the case that Clyburn, 78, was not up to or interested in the task.

But the challenge upset backers of Clyburn, a former Congressional Black Caucus chairman and a revered figure among the caucus’s roughly 50 African Americans — who bristled at the notion that the caucus’s top tier could be all white.

“Out of the three of the leadership positions, he is the only one with announced opposition,” Rep. Cedric L. Richmond, the current Congressional Black Caucus chair, said last week. “I just think it is offensive and insulting.”

Devi Lockwood in Amesbury, Mass., contributed to this report.