Atrophy of the medial temporal lobe (MTL) occurs with aging, resulting in impaired episodic memory. Aerobic fitness is positively correlated with total hippocampal volume, a heavily studied memory-critical region within the MTL. However, research on associations between sedentary behavior and MTL subregion integrity is limited. Here we explore associations between thickness of the MTL and its subregions (namely CA1, CA23DG, fusiform gyrus, subiculum, parahippocampal, perirhinal and entorhinal cortex,), physical activity, and sedentary behavior. We assessed 35 non-demented middle-aged and older adults (25 women, 10 men; 45–75 years) using the International Physical Activity Questionnaire for older adults, which quantifies physical activity levels in MET-equivalent units and asks about the average number of hours spent sitting per day. All participants had high resolution MRI scans performed on a Siemens Allegra 3T MRI scanner, which allows for detailed investigation of the MTL. Controlling for age, total MTL thickness correlated inversely with hours of sitting/day (r = -0.37, p = 0.03). In MTL subregion analysis, parahippocampal (r = -0.45, p = 0.007), entorhinal (r = -0.33, p = 0.05) cortical and subiculum (r = -0.36, p = .04) thicknesses correlated inversely with hours of sitting/day. No significant correlations were observed between physical activity levels and MTL thickness. Though preliminary, our results suggest that more sedentary non-demented individuals have less MTL thickness. Future studies should include longitudinal analyses and explore mechanisms, as well as the efficacy of decreasing sedentary behaviors to reverse this association.