O'Brien said in a statement that "we were never given that chance" to succeed.
"After only seven months, with my Tonight Show in its infancy, NBC has decided to react to their terrible difficulties in prime time by making a change in their long-established late-night schedule," he said.
"I cannot participate in what I honestly believe is [the Tonight Show's] destruction. Some people will make the argument that with DVRs and the Internet a time slot doesn't matter," O'Brien continued. "But with the Tonight Show, I believe nothing could matter more."
Despite his refusal to move the show back a half-hour, O'Brien says he has not received any other offers and remains uncertain about his future. "My hope is that NBC and I can resolve this quickly so that my staff, crew, and I can do a show we can be proud of, for a company that values our work."
The statement followed O'Brien's remarks about the late-night shakeup during his Tonight Show monologue Monday night, when he sounded like a disgruntled employee.
"Everybody wants to know what my plans are," O'Brien said. "Everyone's asking me. All I can say is I plan to keep putting on a great show night after night while stealing as many office supplies as humanly possible. I'm going to rob this place blind. I got 10 cartridges of toner shoved in my pants right now — just jammed down there."
At a Television Critics Association panel Tuesday, ABC Entertainment Group President Steve McPherson said his network had "no plans" to change its late-night lineup featuring Jimmy Kimmel Live! "We love Jimmy," he said.
NBC declined to comment but said a new installment of The Tonight Show would air. Zachary Levi and Roseanne Cash, two of Tuesday's scheduled guests, also confirmed for TVGuide.com that O'Brien was going forward with a new episode.
Fox declined to elaborate on comments made Monday when Fox Entertainment President Kevin Reilly said there had been no formal discussions with O'Brien, but added: "I love Conan personally and professionally."
NBC issued a blanket no comment regarding O'Brien's sentiments.
On the Tonight Show Tuesday night, O'Brien opened with talking about his nearing availability to work children's birthday parties before taking aim at the network behind his professional frustrations. and describing NBC's new slogan as "no longer just screwing up prime time."
"NBC says they're planning to have the late-night situation worked out before the Winter Olympics start. ... And trust me, when NBC says something — you can take that to the bank!"