O'Brien accepted the award for lifetime achievement in comedy Sunday night while acknowledging the backstage turmoil that hangs over the future of the cultural centre in Washington.
O'Brien, 61, was named the 26th recipient of the Mark Twain Prize in mid-January, about three weeks before President Donald Trump upended the Kennedy Center by ousting longtime president Deborah Rutter and board chairman David Rubenstein.
Trump dismissed the board of trustees and replaced them with loyalists, who then elected him as chairman.
On Sunday, O'Brien specifically thanked Rubenstein and Rutter — drawing an extended round of applause — and also Kennedy Center staffers who, he said, were "worried about what the future might bring".
He said the example of Mark Twain's own life and career was particularly resonant at this moment in American history.
"Twain hated bullies ... He punched up, not down, and he deeply empathised with the weak," O'Brien said.
"Twain loved America, but he knew it was deeply flawed."
The uncertain fate of the Kennedy Center itself was woven through the multiple tributes and testimonials to O'Brien.
"I think it would be insane not to address the elephant in the room," comic Nikki Glaser said on the red carpet before the ceremony.
"It's in the air tonight. This night is about Conan, but it can be both."
Once the festivities began, Stephen Colbert joked that the Kennedy Center had announced two new board members: Bashar Assad, the ousted president of Syria, and Skeletor, a fictional supervillain.
John Mulaney quipped that the entire building would soon be renamed "the Roy Cohn Pavilion" after one of Trump's mentors.
Trump, in announcing the Kennedy Center changes, posted on social media that those who were dismissed "do not share our Vision for a Golden Age in Arts and Culture".
Trump has spoken about wanting to book more Broadway shows there and floated the idea of granting Kennedy Center Honors status to actor Sylvester Stallone and singer-songwriter Paul Anka.
In the wake of Trump's takeover of the Kennedy Center, several artists, including the producers of Hamilton and actress and writer Issa Rae, announced they were cancelling appearances at the venue.
