The consumption of calcium, vitamin D, and dairy products may be associated with a reduced risk of type 2 diabetes mellitus. However, whether this reduction is due to calcium, vitamin D, or other components of dairy products is not clear. We examined intakes of total calcium and vitamin D, and plasma concentrations of 25 hydroxyvitamin D [25(OH)D] in relation to fasting plasma concentrations of C-peptide in 2 cross-sectional studies among healthy men from the Health Professionals Follow-up Study and among healthy women from the Nurses' Health Study I. Intake of total calcium was modestly inversely associated with C-peptide concentration in women (P-trend = 0.05); however, the inverse association was not significant in men (P = 0.7). Concentrations of C-peptide were 20% lower among men who had plasma concentrations of 25(OH)D in the highest quartile compared with those in the lowest quartile (P-trend = 0.08); there was no association in women (P = 0.3). The inverse association between calcium intake and the plasma C-peptide concentration was stronger in hypertensive individuals of both sexes. The difference in the C-peptide concentrations between extreme quartiles of calcium intake was 17% in men and 20% in women. Plasma concentrations of C-peptide for the combination of the highest tertiles of calcium intake and plasma 25(OH)D compared with the opposite extreme were 35% lower (P = 0.03) in men and 12% lower (P = 0.01) in women. The results suggest that calcium intake or systemic vitamin D status, after adjustment for intake of dairy products, is associated with decreased insulin secretion.