Objective: The aim of this study is to quantify associations between objectively measured sedentary time and markers of insulin sensitivity by considering allocation into light-intensity physical activity or moderate- to vigorous-intensity physical activity (MVPA). Methods: Participants with an increased risk of impaired glucose regulation (IGR) were recruited (Leicestershire, United Kingdom, 2010-2011). Sedentary, light-intensity physical activity and MVPA time were measured using accelerometers. Fasting and 2-hour post-challenge insulin and glucose were assessed; insulin sensitivity was calculated by HOMA-IS and Matsuda-ISI. Isotemporal substitution regression models were used. Data were analysed in 2014. Results: 508 participants were included (average age. =. 65. years, female. =. 34%). Reallocating 30. min of sedentary time into light-intensity physical activity was associated a 5% (95% CI 1, 9%; p. =. 0.024) difference in Matsuda-ISI after adjustment for measured confounding variables. Reallocation into MVPA was associated with a 15% (7, 25%; p. <. 0.001) difference in HOMA-IS and 18% (8, 28%; p. <. 0.001) difference in Matsuda-ISI. Results for light-intensity physical activity were modified by IGR status with stronger associations seen in those with IGR. Conclusions: Reallocating sedentary time into light-intensity physical activity or MVPA was associated with differences in insulin sensitivity, with stronger and more consistent associations seen for MVPA.