Barlow attended high school in Westfield, Massachusetts, where he met Scott Helland. The two formed the Massachusetts-based hardcore punk band Deep Wound. J Mascis joined the band after answering their ad for a "drummer wanted to play really fast". After becoming disillusioned with the constraints of hardcore, Deep Wound broke up in 1984. Mascis and Barlow reunited to form Dinosaur (later Dinosaur Jr.) later that year. Throughout its early existence, however, Mascis and Barlow had frequent personality conflicts and after the release of their third album Bug in 1988 and the initial supporting tour, Barlow was kicked out of the band.
In 2005, Barlow rejoined the band alongside the original drummer, Murph. Since then, the band has reissued its first three records, toured extensively worldwide and released three new records, Beyond, Farm and I Bet on Sky.
After his dismissal from Dinosaur Jr., Barlow turned his attention to his band Sebadoh, which he had formed several years earlier with multi-instrumentalist Eric Gaffney. The project featured lo-fidelity recording techniques and combined Barlow's introspective, confessional songwriting with Gaffney's discordant noise collages. Bassist and songwriter Jason Loewenstein was later added to the line-up. Sebadoh's early releases include The Freed Man (1989) and Weed Forestin' (1990), the latter of which was originally self-released under the name Sentridoh in 1987. Both albums were officially released by Homestead Records, as was the band's third album, Sebadoh III (1991), which helped establish the "lo-fi" subgenre and became a defining album of 90s indie rock. The band released several studio-recorded albums on Sub Pop Records throughout the 1990s.
As Sebadoh grew in popularity and critical acclaim, Barlow continued work on the Sentridoh side project, which featured mostly home-recorded material similar to his output on the first three Sebadoh albums, but often recorded solo and with a less consistent sound quality. Sentridoh released a trio of cassette-only albums on Shrimper Records in the early 1990s, with the highlights later being collected on CD and vinyl compilations like Winning Losers: A Collection of Home Recordings 89–93 (1994) and Another Collection of Home Recordings (1994). In 1993, Sentridoh released a popular single for the song "Losercore," on the label Smells Like Records founded by Steve Shelley of the American rock band, Sonic Youth. Barlow has called it "the most finely executed of all my releases" thanks to Shelley, who "made sure this looked and sounded great." Numerous other Sentridoh releases on a variety of record labels followed, including releases on his own Loobiecore label.
In 1994, Barlow formed the Folk Implosion with singer-songwriter John Davis. The band released several singles and EPs, and in 1995 reached the Top 40 with the song "Natural One", from the soundtrack to the film Kids by Larry Clark. It remains Barlow's biggest commercial hit. The soundtrack itself was curated by Barlow, and also included previously released songs by Sebadoh, Folk Implosion, Slint and Daniel Johnston.
After the release of Sebadoh's eighth album, The Sebadoh, in 1999, the band went on hiatus and its members went on to pursue other projects. Barlow continued to work with the Folk Implosion, releasing One Part Lullaby in 1999. Barlow took a break from the Folk Implosion in 2000 to collaborate on the album Subsonic 6 with Belgian musician, Rudy Trouvé. In 2003, Barlow released The New Folk Implosion featuring Imaad Wasif on guitar and Sebadoh drummer Russ Pollard on drums. The same year, he appeared as a musician in the film Laurel Canyon. In the spring of 2004, Barlow briefly reunited with Loewenstein for the "Turbo Acoustic" Sebadoh tour. During this tour, he also reunited with J Mascis for a performance of the song "Video Prick" with former Deep Wound vocalist Charlie Nakajima. This performance led to a full-fledged Dinosaur Jr. reunion in 2005, with original members Barlow, Mascis and Murph performing "The Lung" on The Late Late Show with Craig Ferguson on April 15, 2005, and a show at Spaceland in Los Angeles the following night. The band then played well-received tours of the U.S. and Europe throughout the rest of the year, and in 2006 headed to Japan, Australia and New Zealand. In 2007, Barlow reunited with Loewenstein and Gaffney to perform the first Sebadoh shows with the "classic" lineup in 14 years. In June 2013, Sebadoh released their first new music in 14 years; the music was released as an EP precursor to their new album, "Defend Yourself," which was released in September 2013. Both of the new EP and LP albums were released through Joyful Noise Recordings.
Barlow released the first album under his own name, Emoh, in January 2005 on Merge Records. It featured long-time collaborators such as Sebadoh members Jason Loewenstein and Russ Pollard and Lou's sister Abby Barlow, and featured a higher production value than many of his previous solo releases. Jason Crock of Pitchfork called it "the most consistently strong record he's released since The Folk Implosion's One Part Lullaby" and wrote that "even if much of it was recorded at home, Emoh's 14 unassuming folk songs sound like they were created in a professional setting." In November 2005, Barlow toured the Iberian Peninsula to promote the album.