OBJECTIVE: We examined the associations of physical activity with fasting plasma glucose (FPG) and with 2-h postload plasma glucose (2-h PG) in men and women with low, moderate, and high waist circumference. RESEARCH DESIGN AND METHODS: The Australian Diabetes, Obesity and Lifestyle (AusDiab) study provided data on a population-based cross-sectional sample of 4,108 men and 5,106 women aged >or=25 years without known diabetes or health conditions that could affect physical activity. FPG and 2-h PG were obtained from an oral glucose tolerance test. Self-reported physical activity level was defined according to the current public health guidelines as active (>or=150 min/week across five or more sessions) or inactive (<150 min/week and/or less than five sessions). Sex-specific quintiles of physical activity time were used to ascertain dose response. RESULTS: Being physically active and total physical activity time were independently and negatively associated with 2-h PG. When physical activity level was considered within each waist circumference category, 2-h PG was significantly lower in active high-waist circumference women (beta -0.30 [95% CI -0.59 to -0.01], P = 0.044) and active low-waist circumference men (beta -0.25 [-0.49 to -0.02], P = 0.036) compared with their inactive counterparts. Considered across physical activity and waist circumference categories, 2-h PG levels were not significantly different between active moderate-waist circumference participants and active low-waist circumference participants. Associations between physical activity and FPG were nonsignificant. CONCLUSIONS: There are important differences between 2-h PG and FPG related to physical activity. It appears that 2-h PG is more sensitive to the beneficial effects of physical activity, and these benefits occur across the waist circumference spectrum.