The influence of vitamin D status on insulin secretion and glucose tolerance was studied by a longitudinal design in the rabbit. Intravenous glucose tolerance tests were performed in Dutch rabbits (n = 12) before and after nutritional vitamin D deficiency, characterized by an absence of circulating 25-hydroxyvitamin D3, a 50% decrease in 1,25-dihydroxyvitamin D3, and a 16% decrease in serum calcium concentrations. Glucose-induced insulin secretion was reduced by 41% as early as 2 months after the start of the vitamin D-deficient diet and was associated with an impairment of glucose tolerance. An iv calcium infusion restored the serum calcium concentration of the vitamin D-deficient rabbits (n = 5), but did not improve glucose-mediated insulin secretion. When these animals received a single ip injection of 100 ng 1,25-dihydroxyvitamin D3 10 h before the glucose test, their insulin responses significantly increased. Supplementation with 25-hydroxyvitamin D3 for 2 weeks in another group of rabbits (n = 4) resulted in marked improvement in glucose-stimulated insulin release and glucose tolerance. These results show that vitamin D affects glucose-induced insulin secretion by a mechanism that involves more than its regulating action on serum calcium concentration.