OBJECTIVE To examine the prospective association between objectively measured time spent sedentary and insulin resistance and whether this association is independent of moderate- and vigorous-intensity physical activity (MVPA) and other relevant confounders. RESEARCH DESIGN AND METHODS This was a population-based study (Medical Research Council Ely study) in 376 middle-aged adults (166 men; 210 women) over 5.6 years of follow-up. Physical activity and sedentary time were measured objectively by individually calibrated minute-by-minute heart rate monitoring at both baseline and follow-up. Sedentary time was calculated as the heart rate observations (in minutes) below an individually predetermined threshold (flex heart rate) and expressed as a percentage of total monitored time during waking hours over 4 days. The percentage of time spent above 1.75 × resting heart rate represented MVPA. Fasting plasma insulin was used as a surrogate measure of insulin resistance. RESULTS Time spent sedentary at baseline was significantly and positively associated with log fasting insulin at follow-up (β = 0.003, 95% CI 0.0006–0.006, P = 0.015) independent of baseline age, sex, fat mass, fasting insulin, smoking status, and follow-up time. After further adjustment for MVPA, this association was somewhat strengthened (β = 0.004, 95% CI 0.0009–0.006, P = 0.009). CONCLUSIONS Time spent sedentary predicts higher levels of fasting insulin independent of the amount of time spent at moderate- and vigorous-intensity activity levels. This highlights the importance of reducing sedentary time in order to improve metabolic health, possibly in addition to the benefits associated with a physically active lifestyle.