Aims/hypothesis Low serum 25-hydroxyvitamin D [25 (OH)D] concentration may increase risk of insulinrequiring diabetes.Methods A nested case-control study was performed using serum collected during 2002-2008 from military service members. One thousand subjects subsequently developed insulin-requiring diabetes. A healthy control was individually matched to each case on blood-draw date (±2 days), age (±3 months), length of service (±30 days) and sex. The median elapsed time between serum collection and first diagnosis of diabetes was 1 year (range 1 month to 10 years). Statistical analysis used matched pairs and conditional logistic regression. Results ORs for insulin-requiring diabetes by quintile of serum 25(OH)D, from lowest to highest, were 3.5 (95% CI 2.0, 6.0), 2.5 (1.5, 4.2), 0.8 (0.4, 1.4), 1.1 (0.6, 2.8) and 1.0 (reference) (p trend <0.001). The quintiles (based on fifths using serum 25(OH)D concentration in the controls) of serum 25(OH)D in nmol/l, were <43 (median 28), 43-59 (median 52), 60-77 (median 70), 78-99 (median 88) and ≥100 (median 128). Conclusions/interpretation Individuals with lower serum 25(OH)D concentrations had higher risk of insulinrequiring diabetes than those with higher concentrations. A 3.5-fold lower risk was associated with a serum 25 (OH)D concentration ≥60 nmol/l.