The effects of fat content in the hypocaloric diet on whole body glucose oxidation and adipocyte glucose transport were investigated in two animal-feeding experiments. Diet-induced obese rats were food restricted to 75% of their previous energy intakes with either a high (45% by calorie) or a low (12% by calorie) corn oil diet for 9 wk (experiment 1) or 10 days (experiment 2). The losses of body weight (P < 0.05) and adipose depot weight (P < 0.05) were less in the 45% compared with the 12% fat group. During the dynamic phase of weight loss (day 10 of food restriction), plasma glucose and insulin concentrations were higher (P < 0.05) in the 45% than those in the 12% fat group. Whole body carbohydrate oxidation rate in response to an oral load of glucose was increased (P < 0.001) by food restriction in both dietary groups; however, carbohydrate oxidation rates were lower (P < 0.01) in the 45% than in the 12% fat-fed rats during the weight loss period. Adipocyte glucose transport was greater (P < 0.02) in the 45% than in the 12% fat group in an intra-abdominal adipose depot but not in subcutaneous fat. These data suggest that dietary fat content modifies whole body glucose oxidation and intra-abdominal adipocyte glucose uptake during weight loss.