OBJECTIVE: We examined the associations of objectively measured sedentary time, light-intensity physical activity, and moderate- to vigorous-intensity activity with fasting and 2-h postchallenge plasma glucose in Australian adults. RESEARCH DESIGN AND METHODS: A total of 67 men and 106 women (mean age +/- SD 53.3 +/- 11.9 years) without diagnosed diabetes were recruited from the 2004-2005 Australian Diabetes, Obesity, and Lifestyle (AusDiab) study. Physical activity was measured by Actigraph accelerometers worn during waking hours for 7 consecutive days and summarized as sedentary time (accelerometer counts/min <100; average hours/day), light-intensity (counts/min 100-1951), and moderate- to vigorous-intensity (counts/min > or =1,952). An oral glucose tolerance test was used to ascertain 2-h plasma glucose and fasting plasma glucose. RESULTS: After adjustment for confounders (including waist circumference), sedentary time was positively associated with 2-h plasma glucose (b = 0.29, 95% CI 0.11-0.48, P = 0.002); light-intensity activity time (b = -0.25, -0.45 to -0.06, P = 0.012) and moderate- to vigorous-intensity activity time (b = -1.07, -1.77 to -0.37, P = 0.003) were negatively associated. Light-intensity activity remained significantly associated with 2-h plasma glucose following further adjustment for moderate- to vigorous-intensity activity (b = -0.22, -0.42 to -0.03, P = 0.023). Associations of all activity measures with fasting plasma glucose were nonsignificant (P > 0.05). CONCLUSIONS: These data provide the first objective evidence that light-intensity physical activity is beneficially associated with blood glucose and that sedentary time is unfavorably associated with blood glucose. These objective data support previous findings from studies using self-report measures, and suggest that substituting light-intensity activity for television viewing or other sedentary time may be a practical and achievable preventive strategy to reduce the risk of type 2 diabetes and cardiovascular disease.