The locations and habitat features of freshwater holding areas and the timing of fall migration are undocumented for Gulf sturgeon in the Pascagoula River drainage, Mississippi. Our objectives were to identify and characterize holding areas for Gulf sturgeon (Acipenser oxyrinchus desotoi), document their summer and fall movement patterns, and determine migration cues. To do this we captured, radio-tagged, and monitored movement of Gulf sturgeon in the Pascagoula River drainage and analyzed these data using geographic information systems. From May to November Gulf sturgeon congregate in a holding area in the lower portion of the Pascagoula River and Big Black Creek [river kilometers (rkm) 57–68] and near Cumbest Bluff (rkm 40), before they return to the Gulf of Mexico. While in the holding area, Gulf sturgeon were typically found in deep locations, either in or downstream from river bends. As found in other rivers, Gulf sturgeon in the Pascagoula River showed little movement within the holding area and often stayed within a single river bend; although we observed local movements by some individuals (under 10 rkm). In the Pascagoula River, Gulf sturgeon initiated their migration out of fresh water from late-September to mid-October, coincident with shorter day length, falling water temperature (mean = 23.7°C, range 21–26°C), and elevated river flow. Our work demonstrates that the lower Pascagoula River serves as a vital area for Gulf sturgeon.