The decision has thrust Booker, a New Jersey Democrat and fixture on lists of potential 2020 presidential candidates, into the limelight.
Republicans quickly accused Booker of seeking to improve his own political prospects.
Democrats made clear Booker is on his own.
"It's not for me to approve or disapprove. He's asked to testify and his message is whatever he wants it to be," said Sen. Dick Durbin of Illinois, the No. 2-ranking Democrat.
Now, liberals searching for new leadership within their party -- and a strong voice of opposition in the Trump era -- will be watching Booker closely to see if he meets the moment he created for himself.
That one sitting senator will testify against another is historic for a chamber where decorum rules the day and senators do not even address each other directly on the floor.
But that it's Booker, in particular, raised eyebrows. He's known as a bipartisan leader on criminal justice reform and has worked to pass a bill with hard-line conservative Sen. Ted Cruz of Texas.
After Booker was added to the list of those testifying on the second day of Sessions' confirmation hearing, Sen. Tom Cotton, R-Arkansas, attacked Booker, accusing him on Facebook of a "disgraceful breach of custom."
"This hearing simply offers a platform for his presidential aspirations. Senator Booker is better than that, and he knows better," Cotton wrote.
The Republican National Committee highlighted Booker's comments that he felt "blessed and honored to have partnered with Sen. Sessions" in February 2016 to pass legislation honoring voting rights march foot soldiers with the Congressional Gold Medal.
Sen. Tim Scott, a South Carolina Republican who is close to Booker, told CNN that Booker's testimony won't affect their relationship.
"I think Cory's a good guy and is making a decision. It's a very unique decision, obviously; it's never been done before. I think looking back 20 years from now, we'll understand the impact of his decision to do so," Scott said.
"It's a whole lot easier to understand looking back the impact a decision has had on the body, the camaraderie, the necessity of working across the aisle," Scott said. "There's a lot of things that could be impacted by the decision. We'll have to see how he does what he does."
Liberal groups -- particularly the NAACP and pro-LGBT organizations including the Human Rights Campaign -- have made Sessions' nomination their top target, hoping to derail at least one Trump selection.
Sessions' 1986 nomination for a federal judgeship was derailed over accusations of racist comments.
Testifying before the Senate Judiciary Committee Tuesday, Sessions diverged from his prepared remarks to address "head on" the very allegations that helped sink him in 1986, including that he'd joked about not having a problem with the Ku Klux Klan.
"I abhor the Klan and its hateful ideology," Sessions said.
For his part, Booker said Tuesday that he felt a "call to conscience" to oppose Sessions.
"I do not take lightly the decision to testify against a Senate colleague," Booker said. "But the immense powers of the attorney general combined with the deeply troubling views of this nominee is a call to conscience."
Booker told CNN on Tuesday morning shortly before Sessions' hearing started that it was "consequential moment."
"This is one of the more consequential appointments in American history right now given the state of a lot of our challenges we have with our policing, a lot of challenges we have with race relations, gay and lesbian relations," Booker said.
Booker called Sessions' record "concerning in a number of ways," citing his opposition to bipartisan criminal justice reform and immigration reform, criticism of the Voting Rights Act and his "failure to defend the civil rights of women, minorities, and LGBT Americans."