Evidence of autumn spawning of Gulf sturgeon Acipenser oxyrinchus desotoi in the Suwannee River, Florida, was compiled from multiple investigations between 1986 and 2008. Gulf sturgeon are known from egg collections to spawn in the springtime months following immigration into rivers. Evidence of autumn spawning includes multiple captures of sturgeon in September through early November that were ripe (late-development ova; motile sperm) or exhibited just-spawned characteristics, telemetry of fish that made >175 river kilometer upstream excursions to the spawning grounds in September–October, and the capture of a 9.3 cm TL age-0 Gulf sturgeon on 29 November 2000 (which would have been spawned in late September 2000). Analysis of age-at-length data indicates that ca. 20% of the Suwannee River Gulf sturgeon population may be attributable to autumn spawning. However, with the very low sampling effort expended, eggs or early life stages have not yet been captured in the autumn, which would be the conclusive proof of autumn spawning. More sampling, and sampling at previously unknown sites frequented by acoustic telemetry fish, would be required to find eggs.