Casting aside her trademark warmth and brimming eyes, Winfrey wasted no time on niceties, plunging straight into short, direct questions that saw the seven-times Tour de France winner end years of denials.
"Were you a bully?" Winfrey challenged Armstrong, referring to the pressure he put on cycling team-mates to join him in taking performance enhancing substances.
"Tell me, how were you a bully?" she persisted at one point in the first 90-minute televised interview on her OWN cable channel.
Winfrey, 58, pioneered the art of confessional TV during her 25 year-long reign on her daytime chat show, making her comfortable couches the go-to place for celebrities wanting to apologize for indiscretions or unburden their darkest secrets.
But on Thursday's broadcast, bright sofas were replaced with two hard-backed chairs, and Armstrong was made to watch video of his past victory speeches and interviews.
"You didn't just deny it. You brazenly and defiantly denied it," she told Armstrong over his years of repeated lies.
"This is too late. It is too late for probably most people and that is my fault," he conceded. "I view this situation as one big lie."
"You are suing people, and knowing they are telling the truth. What is that?" she later asked of Armstrong's legal actions against journalists, friends and fellow team members who spoke out over the years.
Armstrong, 41, dressed in an open-necked blue shirt and dark jacket, may have been expecting a different reception when he chose Winfrey as his confessor, rather than holding a news conference or issuing a statement.
"Oprah is so overbrimming with humanity that the person being interviewed hopes that a bit of it will rub off on them," said Robert Thompson, professor of pop culture at Syracuse University, before the broadcast.
"Part of sitting down and doing an interview and confessing to another person and being asked questions is almost like you are starting to pay a penalty.
"The idea is that Oprah will do that, not necessarily in a softball way, but in a gentle way. Oprah is not going to hit you on the head," Thompson told Reuters.
The smiles were few and far between on Thursday, as Winfrey dug deep for answers without ever raising her voice.
"I can't reconcile what you were thinking when you did this?" she said, after playing video of Armstrong's 2005 Tour de France victory speech in which he dismissed critics as cynics.
A second hour of the interview, taped earlier this week in Texas, will be shown on Friday.
"Was it just you being your cocky, arrogant, jerk self?" Winfrey asks Armstrong in an excerpt of Friday's one-hour session.
Although Winfrey's influence has waned since she ended "The Oprah Winfrey Show" in 2011 to launch her OWN cable channel, she has proved she can still pull in the big names, if not the huge audiences she enjoyed when her show was broadcast in 140 nations.
Landing Armstrong was arguably Oprah's finest moment since she coaxed pop singer Michael Jackson out of 14 years of TV interview silence for a 1993 broadcast watched by an estimated 90 million people worldwide.
For much of Thursday, the OWN cable channel ran a countdown clock during regular programming ahead of "Oprah and Lance Armstrong: The Worldwide Exclusive."
The interview was also watched online by viewers from as far afield as Britain, Singapore, South Korea and Brazil, according to comments on Oprah.com.
Winfrey herself termed the sit-down with Armstrong "certainly the biggest interview I have ever done in terms of exposure."
What may prove a huge PR boost to Oprah and her OWN TV ratings, may not be so clear-cut for Armstrong.
"I'm not sure what he expects, or what he can achieve by fessing up after so many years. But if Armstrong wants to be talked about, you couldn't do better than doing it with Oprah," said Thompson.