PURPOSE: The effect of sedentary behavior on regional fat deposition, independent of physical activity, remains equivocal. We examined the cross-sectional associations between objectively measured sedentary time and markers of regional fat distribution (heart, liver, visceral, subcutaneous, and total body fat) in a population at a high risk of type 2 diabetes mellitus (T2DM). METHODS: Participants were recruited from primary care to two diabetes prevention programs. Sedentary time (<25 counts per 15 s) was measured using ActiGraph GT3X accelerometers. Heart, liver, visceral, subcutaneous, and total body fat were quantified using magnetic resonance images. Fat volumes were calculated by multiplying the cross-sectional areas of the fat-containing pixels by the slice thickness. The liver fat percentage was measured using a representative region of interest created in the right lobe of the liver, avoiding the main portal veins. Linear regression models examined the association of sedentary time with markers of regional fat deposition. RESULTS: Sixty-six participants (age, 47.9 ± 16.2 yr; male, 50.0%) were included. After adjustment for several covariates, including glycemia, whole-body fat, and moderate-to-vigorous physical activity, each 30 min of sedentary time was associated with 15.7 cm higher heart fat (P = 0.008), 1.2% higher liver fat (P = 0.026), and 183.7 cm higher visceral fat (P = 0.039). CONCLUSIONS: This study provides new evidence suggesting that objectively measured sedentary behavior may have an independent association with heart, liver, and visceral fat in individuals at a high risk of T2DM.