Epidemiological studies report inverse associations between blood vitamin D, as measured by 25-hydroxyvitamin D [25(OH)D] concentrations, and insulin resistance (IR) among predominantly overweight individuals. In a cross-sectional survey of 5 Cree communities in Quebec, Canada, we determined if 25(OH)D is associated with IR and β-cell function in a largely obese, ethnic minority at high risk of developing type 2 diabetes. A total of 510 participants (≥18 y) without type 1 or type 2 diabetes, assessed for serum 25(OH)D, fasting plasma glucose and insulin, and anthropometric and lifestyle variables, were included in the analyses. Multivariable linear regressions adjusted for covariates were performed for homeostasis model assessment of IR (HOMA-IR) and β-cell function (HOMA-B) in relation to serum 25(OH)D. Serum 25(OH)D (per 10 nmol/L increment) was inversely associated with HOMA-IR (β = -0.005; SE = 0.002; P = 0.004) and HOMA-B (β = -0.004; SE = 0.002; P = 0.006) in models adjusted for age, sex, physical activity, education, alcohol consumption, and smoking. When further adjusted for BMI, associations were no longer significant for either HOMA-IR (β = 0.001, SE = 0.002, P = 0.572) or HOMA-B (β = 0.001, SE = 0.001, P = 0.498). The modest inverse associations between 25(OH)D and IR reported previously were not observed in this population after adjusting for adiposity. Future longitudinal studies investigating the interrelationship among 25(OH)D, adiposity, and the risk of developing metabolic syndrome and type 2 diabetes are warranted.