He played regularly around the Southeastern US with The Hilltops, a band based in Oxford, Mississippi that included his twin sister Laurie Stirratt and her husband Cary Hudson. During this time he met and befriended the band Uncle Tupelo and supported them on tours of the East and Midwest.
After the breakup of The Hilltops in 1990 Stirratt recorded a record under the name The Gimmecaps and briefly joined Lafayette, LA band The Bluerunners before joining Uncle Tupelo in 1992 as bassist/guitarist on their last album Anodyne.
After the breakup of Uncle Tupelo Stirratt rejoined Jeff Tweedy, Ken Coomer and Max Johnston to found Wilco in 1994. Since the founding of Wilco, Stirratt and Tweedy are the only members to contribute to all Wilco releases. Stirratt also joined Wilco members Jay Bennett and Ken Coomer to form Courtesy Move, an early Wilco side project that recorded an album in late 1996 that was never released.
Stirratt formed The Autumn Defense in 2000 with friend and fellow New Orleanian Pat Sansone. In 2001 they recorded their debut The Green Hour in New Orleans and Nashville with former Wilco members Bob Egan and Ken Coomer, releasing the album on Stirratt’s own Broadmoor Records.
The Autumn Defense released Circles in 2003 with Greg "G. Wiz" Wieczorek on drums and Nashville-based bassist Brad Jones. When Sansone joined Wilco in 2004, The Autumn Defense had more time to collaborate, resulting in their third, self-titled album (2006). The Autumn Defense focused on Sansone's orchestral production style.
Stirratt and his sister, Laurie, also recorded a duet "Laurie & John" on the folk-rock album Arabella (2003). This was the twins' first recorded material in 12 years.