In order to clarify the role of vitamin D (D) in regulating insulin secretion, we studied the effect of long term (10 days) and short term (3 days) supplementation with D and/or calcium (Ca) on insulin secretion from the isolated, perfused pancreas of D- and Ca-deficient rats. The influence of the nutritional state induced by D deficiency was also evaluated. The long term supplementation of either D, Ca, or both restored the body weight and improved insulin secretion induced by high glucose concentration to the same extent; thus, no significant difference in insulin secretion was found between the D-only-supplemented group and the Ca-only-supplemented group. When the insulin secretion was compared in D-deplete vs. D-replete rats given the same amount of Ca, insulin secretion was significantly higher in D-replete animals, although plasma Ca levels were also higher. In short term experiments, insulin release was significantly augmented to a similar extent in D- or Ca-replete rats as compared with D- and Ca-deficient rats, despite no significant change in body weight. In a separate experiment, the pancreas from D-deficient rats was perfused with or without 1,25-dihydroxyvitamin D3 [1,25-OH)2D3] to observe its acute effect on insulin release. The perfusion with 1,25-(OH)2D3 did not affect insulin release. This result suggests that impaired insulin secretion in D-deficient rats is caused by a decrease in Ca in the body fluid and possibly by the lack of D effect on the pancreas.