OBJECTIVE To evaluate whether sedentary time (ST) and/or sedentary behavior patterns are related to incident diabetes in the U.S.’s oldest age-groups. RESEARCH DESIGN AND METHODS Women without physician-diagnosed diabetes (n = 4,839, mean ± SD age = 79 ± 7 years) wore accelerometers for ≥4 days and were followed up to 6 years for self-reported newly diagnosed diabetes requiring treatment with medications. Hazard ratios (HRs) for incident diabetes were estimated across quartiles of accelerometer-measured ST and mean bout duration with use of Cox proportional hazards models. We conducted isotemporal substitution analyses using Cox regression and tested associations with risk for diabetes after statistically replacing ST with light physical activity (PA) or moderate-to-vigorous PA (MVPA) and after replacing light PA with MVPA. RESULTS During 20,949 person-years, 342 diabetes cases were identified. Women in ST quartile (Q)2, Q3, and Q4 (vs. Q1) had incident diabetes HR 1.20 (95% CI 0.87–1.65), 1.33 (0.97–1.82), and 1.21 (0.86–1.70); Ptrend = 0.04. Respective HRs following additional adjustment for BMI and MVPA were 1.04 (95% CI 0.74–1.47), 1.04 (0.72–1.50), and 0.85 (0.56–1.29); Ptrend = 0.90. Fully adjusted isotemporal substitution results indicated that each 30 min of ST replaced with MVPA (but not light PA) was associated with 15% lower risk for diabetes (HR 0.85 [95% CI 0.75–0.96]; P = 0.01); the HR for replacing 30 min of light PA with MVPA was 0.85 (95% CI 0.73–0.98); P = 0.03. Mean bout duration was not associated with incident diabetes. CONCLUSIONS Statistically replacing ST or light PA with MVPA was associated with lower diabetes risk in older women. While reducing ST is important for several health outcomes, results indicate that to reduce diabetes risk among older adults, the primary public health focus should be on increasing MVPA.