OBJECTIVE - To determine the association between serum 25-hydroxyvitamin D (250HD) and diabetes risk and whether it varies by ethnicity. RESEARCH DESIGN AND METHODS - We performed an analysis of data from participants who attended the morning examination of the Third National Health and Nutrition Examination Survey (1988-1994), a cross-sectional survey of a nationally representative sample of the U.S. population. Serum levels of 250HD, which reflect vitamin D status, were available from 6,228 people (2,766 non-Hispanic whites, 1,736 non-Hispanic blacks, and 1,726 Mexican Americans) aged greater than or equal to20 years With fasting and/or 2-h plasma glucose and serum insulin measurements. RESULTS - Adjusting for sex, age, BMI, leisure activity, and quarter of year, ethnicity-specific odds ratios (ORs) for diabetes (fasting glucose greater than or equal to7.0 mmol/l) varied inversely across quartiles of 250HD in a dose-dependent pattern (OR 0.25 [95% CI 0.11-0.60] for non-Hispanic whites and 0.17 [0.08-0.37] for Mexican Americans) in the highest vitamin D quartile (250HD greater than or equal to81.0 nmol/l) compared with the lowest 250HD (less than or equal to43.9 nmol/l). This inverse association was not observed in non-Hispanic blacks. Homeostasis model assessment of insulin resistance (log,) was inversely associated with serum 250HD in Mexican Americans (P = 0.0024) and non-Hispanic whites (P = 0.058) but not non-Hispanic blacks (P = 0.93), adjusting for confounders. CONCLUSIONS - These results show an inverse association between vitamin D status and diabetes, possibly involving insulin resistance, in non-Hispanic whites and Mexican Americans. The lack of an inverse association in non-Hispanic blacks may reflect decreased sensitivity to vitamin D and/or related hormones such as the parathyroid hormone.