Outside of music, Isbell has appeared in roles in the television series Squidbillies and Billions, as well as the films Deadwood: The Movie and Killers of the Flower Moon.

His grandfather and uncle taught him to play various instruments,[11] including the mandolin when he was six years old, as it was easier for him to grip as a small child.

By this time, Patterson Hood and his future Drive-By Truckers co-founder, Mike Cooley, were older and had moved out of town.

[2] Isbell submitted demos and eventually got a publishing deal with FAME Studios of Muscle Shoals, Alabama, when he was 21.

Co-founder Patterson Hood recalls that he met Isbell through Dick Cooper, a mutual friend from Muscle Shoals.

[21] Hood invited Isbell to join the Drive-By Truckers after he sat in with the group at an acoustic house party when guitarist Rob Malone did not show up.

[22] Isbell recorded and contributed many songs to the Drive-By Truckers for their next three albums, 2003's Decoration Day, 2004's The Dirty South, and 2006's A Blessing and a Curse.

The title track of Decoration Day was revealed by Isbell in the 2014 Live from Lincoln Center concert to be a true story about his family members.

[10] On June 15, 2014, Isbell teamed with Hood and Mike Cooley for a benefit at the Shoals Theater in Florence, Alabama.

Produced by Dave Cobb and featuring accompanying vocals by Kim Richey and Isbell's wife, Amanda Shires, Southeastern received overwhelmingly positive critical reviews, earning a score of 87[30] on Metacritic.

[36] During the summer of 2015, Isbell was on a North American tour to promote the album, with four consecutive sold-out nights at the Ryman Auditorium in Nashville at the end of October.

[37][38] In April 2016, Isbell appeared on the BBC live-music show Later With Jools Holland, singing "The Life You Chose", one of the tracks from Something More Than Free.

Isbell said that compared to Southeastern, Something More Than Free has a feeling of celebration,[39][40] which reflects his upcoming fatherhood and a forward-facing momentum.

[51] Isbell also contributed the ballad "Maybe It's Time" to the soundtrack of the 2018 film A Star Is Born, where it was performed by actor Bradley Cooper's character, Jackson Maine.

[53] Isbell once again worked with producer Dave Cobb on the album which featured guest vocals by David Crosby as well as Jay Buchanan of Rival Sons.

[55] The album, entitled Georgia Blue, was formally announced on September 14, 2021, with release dates of October 15 for the digital version and November 26 for CD and vinyl.

The album, produced by Isbell, included the singles, "Strawberry Woman," "Cast Iron Skillet," and "Miles."

Isbell's first acting role came in 2016 when he guest starred in the animated TV series Squidbillies, providing the voice of pastor Kyle Nubbins.

The show has featured other Americana singers in cameo roles, including Elizabeth Cook, Todd Snider, and the Drive-By Truckers, among others.

[61] In 2021, Isbell was cast in Martin Scorsese's epic Western crime drama film Killers of the Flower Moon (2023) as Bill Smith, a victim of the Osage Indian murders.

[63] Isbell has spoken about the importance of his northern Alabama roots: "I definitely don't feel like I would be the musician that I am, or the type of songwriter, had I not come from that particular place," he said in 2012.

"[10] In addition to citing Neil Young as a big influence, Isbell is a fan of singer-songwriter Ben Howard and guitarist Blake Mills.

[9] Isbell was previously married to Shonna Tucker, a fellow musician from the Muscle Shoals community and a former bass player for Drive-By Truckers.

Isbell has a tattoo on the inside of his left arm with a quotation from the lyrics of the Bob Dylan song "Boots of Spanish Leather": "Just carry yourself back to me unspoiled, from across that lonesome ocean.

[77][78][79] In one instance of the former, Isbell posted the following to Twitter on the day of the 2019 Dayton shooting: "If you're on here arguing the definition of 'assault weapon' today you are part of the problem.

"[80] A Twitter user by the name of William McNabb responded to Isbell, asking what he as a "rural American" should use to kill the "30-50 feral hogs" in his yard.

McNabb's tweet itself soon became an internet meme, with Twitter users widely mocking him for what was perceived to be an absurd hypothetical question that was quizzically worded.