Knowlton took aim and fired two shots with a high-powered rifle from less than 30 feet away, CNN reported.
A third shot was fired and the animal was dead.
It was the end of a saga that began when Knowlton purchased the permit to hunt the animal at a January 2014 auction.
The bull, Knowlton said, was a problem in his own herd. The animal was too old to breed but so aggressive that it had already killed calves, cows and and other male rhinoceroses in a jealous rage.
Prized by poachers, black rhinos are critically endangered; there are fewer than 5,000 of them left on Earth. But the threat to their survival is from outside and within, Knowlton said.
Proponents call it "conservation hunting," the practice of offering hunting opportunities for a fee that can then be used on the conservation effort.
In a statement, the International Union for the Conservation of Nature said the concern over killing a rhinoceros for sport is understandable but confuses illegal poaching with well-managed hunting tourism.
“Well-managed trophy hunting has little to do with poaching, and indeed can be a key tool to help combat it,” the union said. Without it, African conservationists “would not be able to employ the upwards of 3,000 field rangers employed to protect wildlife and enforce regulations.”
Knowlton shot the bull "after a three-day hunt through the bush with government officials on hand to ensure he killed the correct animal," AFP reported.
Asked afterward if he still believed his actions benefited the species, Knowlton responded: "100 percent."
"I'm pretty emotional right now, to be honest," he told CNN. "I felt like from day one it was benefiting the black rhino, and I'll feel like that until the day that I die."
He added: "Being on this hunt, with the amount of criticism it brought and the amount of praise it brought from both sides, I don't think it could have brought more awareness to the black rhino."
Knowlton paid a massive sum of money for the permit from the Namibia Ministry of Environment and Tourism, then found himself on the receiving end of death threats. Tens of thousands of people petitioned the U.S. Fish and Wildlife Service to prevent him from importing the rhinoceros carcass after the hunt.
In its statement announcing the decision, the federal agency noted that hunting specific older bulls that were known to keep cows in the herd from mating with other bulls was necessary to increase the rhino population.
Knowlton let CNN cameras in on the hunt for further vindication.
"At this point, the whole world knows about this hunt and I think it's extremely important that people know it's going down the right way, in the most scientific way that it can possibly happen," he told the network.
He added: "I think people have a problem just with the fact that I like to hunt. ... I want to see the black rhino as abundant as it can be. I believe in the survival of the species."
Still, Knowlton remains the enemy of opponents of conservation hunting.
"I am deeply saddened, disappointed and incredulous that he sees this mission as contributing to the survival of endangered black rhinos," Jeff Flocken, North America regional director for the International Fund for Animal Welfare, said in a statement on Wednesday. "If you pay to take a human life and give to humanitarian causes, it does not make you a humanitarian. And paying money to kill one of the last iconic animals on earth does not make you a conservationist."
As for the rhino Knowlton bagged: Meat from the 3,000 pound animal was taken to a nearby village for food. And Knowlton will import the horns, the hide and body to the U.S. as his hunter's trophy, according to CNN.
"It’s hard to say why hunters value the remains so much — respect, a memorial, the time you had with it, I believe it’s all of that,” Knowlton told The Post months before the hunt. "A hunter’s relationship with wildlife is intimate."
[This post has been update.]