Water depth may be a primary factor that influences aquatic habitat use by Glyptemys insculpta (Wood Turtle). We used radio telemetry to investigate the influence of sex and activity period on water depth used by Wood Turtles to reveal aquatic behavioral patterns with potential habitat-management implications for this imperiled species. We located 42 Wood Turtles (28 females, 14 males) from 3 sites in Iowa biweekly to bimonthly during 2017–2021. We measured water depth at 1784 individual turtle locations and compared turtle depth to the minimum and maximum depths within 10 m of the turtle, and stream depth at mid-channel, to determine if water depth differed between sexes and/ or 5 seasonal activity periods (brumation, pre-nesting, nesting, post-nesting, and pre-brumation). Activity period significantly affected water depth used, and both sexes chose deeper water locations during winter brumation (mean = 110.6 cm) than during the other 4 activity periods. However, sex, and the interaction between sex and activity period, were not significant factors. Both sexes utilized deep water (81–121 cm) most frequently (35.6% of observations) during brumation. Utilization of moderately deep, flowing-water areas during winter may be an adaptation in Wood Turtles that provides protection from thickening river ice and fluctuating water levels while they are largely inactive. Our results provide data that can be used by habitat managers to mitigate the effects of various bridge, roadway, and stream-based construction projects that may influence stream depth and water flow in Wood Turtle habitat.