Holt's first job was a Country and Western station disc jockey, which he held throughout his time at college. In 1981 he was hired by WCBS-TV to be a reporter in New York City. The following year he went to Los Angeles to work for KNXT, but returned to WCBS-TV the following year. In 1986 he moved to WBBM-TV in Chicago where he finally found a home and spent the next 14 years anchoring the evenings news. 

Holt joined MSNBC in 2000 and held various positions including duties with NBC News, NBC Nightly News, Today, and Weekend Today. He assumed the permanent anchor position for NBC Nightly News on June 18, 2015 following the demotion of Brian Williams. This made him the first African-American to solo anchor a weekday network nightly newscast. "We all have to be reminded of where we came from and how we got here and the doors that were opened up.", Holt commented on his privilege. "Certainly, as an African-American, those doors were opened up through a lot of sacrifice. It's reminded me of the shoulders that I stand on, that I had the opportunity to do whatever I wanted in life."

"Around here the nickname for Lester is Iron Pants, because he can sit down and anchor for hours", said MSNBC vice president Mark Effron. Holt agrees. "When there's breaking news, I don't want to leave that chair." "People say they're overworking me. I say that's opportunity", he continued. "You never get bored." He is the current host for NBC's Dateline. 

Holt moderated the presidential candidates' debates in January of 2016 as well as the first presidential debate on September 26, 2016. Then presidential candidate Donald Trump initially said this was "a very unfair system" because "Lester is a Democrat." Holt is, in fact, a registered Republican. He was praised for his role in fact-checking false statements made by both candidates. After the debate, Donald Trump admitted that Holt did "a good job" and journalists on both sides overwhelmingly agreed, stating that Holt "performed admirably." "It was the most difficult thing I've ever done professionally or maybe even personally", he recounted.

His favorable performance allowed Holt to interview President Trump again in May of 2017, which TV news anchor Lawrence O'Donnell called "possibly the most important televised interview ever done." Holt and Trump discussed the firing of FBI director James Comey. Trump later accused NBC of "fudging" the tape in order to paint him in a negative light. He was quoted as saying "And, in fact, when I decided to just do it I said to myself, I said, You know, this Russia thing with Trump and Russia is a made-up story, it's an excuse by the Democrats for having lost an election that they should've won." This contradicted his previous statements which stated Comey's dismissal was based on recommendations by Rosenstein and Attorney General Jeff Sessions and was related to the mishandling of Hillary Clinton's emails. Several reporters came to NBC's defense, stating the entire 13 minute interview had been publicly posted for months.