The spatial and seasonal occupancy by Gulf Sturgeon Acipenser oxyrinchus desotoi was estimated using acoustically tagged fish in the Pascagoula River estuary over a 3-year period (2010-2013) and was based on 131,381 detections. Juveniles spent considerably more time in the estuary than subadults or adults, but that varied by year. Gulf Sturgeon of all size-classes entered the acoustic array earlier when stream discharge was high (2011, 2012) compared with when it was lower (2010). The difference was about 14-28 d earlier for adults and subadults but only 12-17 d for juveniles. Temporally, there were no significant differences in occupancy by year or season for adults or subadults or by year for juvenile Gulf Sturgeon, suggesting repeated patterns across the 3 years of this study. Significant and consistent spatial differences in occupancy occurred by array zone, with juveniles occupying the West zone more than the East and River zones of the acoustic array; however, occupancy in the River zone was greater than in the East zone. We found similar patterns for subadults, with the highest occupancy in the West zone compared with the East zone; use was higher in the River zone than in the East zone but there was no difference between the River and West zones. In contrast, no year, season, or zone patterns were identified for adult Gulf Sturgeon, suggesting annual movement along a defined travel corridor (from the River zone to the West zone to offshore islands), with little time spent in the estuary. Adults appear to move quickly through the system during emigration (fall) and immigration (spring) compared with longer but more variable occupancy for juveniles and subadults. Our long-term data illustrates the importance of habitat connectivity in the recovery of Gulf Sturgeon and enhances earlier work on estuarine zones used during migration periods and estuarine residency; both are important for a better understanding of critical habitat maintenance and the development of a robust recovery plan.