Colbert asked Winfrey about past comments she made, where she explained that she would need a clear sign from God before she would even consider running and that she hasn't yet seen one.
Luckily for Colbert, God himself decided to join the conversation from the roof of New York City's Ed Sullivan Theater, telling the A Wrinkle in Time star that he's a "huge fan."
"I'm sorry about this, Oprah. I apologize. God stops by every once in a while," Colbert told Winfrey, who was suppressing a laugh and clearly surprised by the deity's unexpected appearance. "Can I help you, Lord? I'm kind of the in middle of talking to somebody important."
"I hear thou seeketh a sign? Well, is this clear enough?" the projection of God asked, holding up a cardboard sign that simply read, "Run!"
"Well, all I can say, God, is that now 'run' is now a part of my exercise routine. I can tell you that," she replied.
In an attempt to make things even clearer, God donned a hat and T-shirt bearing her possible campaign slogans.
Winfrey wouldn't budge, however, explaining that she's never imagined herself running for president. "It's not the kind of job you can have without fully devoting yourself to it 100 percent," she said.
"Not really," God shot back. "Have you seen this White House?"
While he couldn't convince her, God did have one final request: "Any chance you could get the Bible into your book club?"
"I'm working on that," Winfrey promised with a smile.
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Oprah Winfrey Joins the '60 Minutes' Family
Earlier in the episode, Winfrey opened up about her work as a special correspondent for 60 Minutes, in which she interviewed two groups of people, one with voters who support President Donald Trump and those who do not, in an effort to examine and explore their ideological differences and similarities.
Following her highly publicized 60 Minutes segment, Trump made headlines when he slammed Winfrey for what he called "biased" interviewing and said he hopes to face off against her if she decided to run for president.
"Just watched a very insecure Oprah Winfrey, who at one point I knew very well, interview a panel of people on 60 Minutes," he tweeted. "The questions were biased and slanted, the facts incorrect. Hope Oprah runs so she can be exposed and defeated just like all of the others!"
"Now I would say, if there is one thing I associate with you, it's insecure," Colbert joked.
Winfrey said she didn't reject the criticism outright but rather tried to consider Trump's remarks and see if they could have held water.
"When I heard that the president had tweeted that, I went back and looked at the tape to see what [he was] talking about," she recalled. "And I didn't get it, but I did examine it. I called up the 60 Minutes producers, I said, 'Hey, is there any validity to this at all?' And we all looked at it and said no."
Before welcoming Winfrey out as his first guest of the night, Colbert had some big news of his own to share with the audience: Former FBI Director James Comey will be joining him as a guest on April 17, marking Comey's first late night TV appearance since being fired.