According to a tweet posted by Isbell's record label on Tuesday, Isbell and the 400 Unit band won't be performing because the venue "was not willing to comply with the band's updated Health and Safety standards."
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The Grammy-Award-winning singer and his band have been requiring audiences to provide proof of vaccination or a negative COVID-19 test.
"If the venue won't allow that, we won't play," Isbell said in a tweet posted on Monday.
Isbell is one of many artists requiring vaccinations and tests.
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Global Citizen announced Tuesday that a 24-hour broadcast of performances across multiple networks and platforms will take place on six continents on September 25.
New York's Central Park will host 60,000 fans who'll dance to artists like Coldplay, Billie Eilish, Jennifer Lopez, Lizzo and Meek Mill, while Ed Sheeran, Doja Cat, H.E.R. and more will join the Peas and 20,000 attendees in Paris.
In order to attend, concertgoers must provide either proof that they are fully vaccinated or present a negative PCR test within 72 hours of the show's start, and every attendee must be masked regardless of vaccination status.
The Associated Press contributed to this report.