Vitamin D is essential for normal insulin secretion in vivo and in vitro. The differential effect of calcium and of the vitamin D endocrine system in the insulin response to secretagogues is still a subject of debate. To study the roles of calcium and the vitamin D system in the in v vitamin D depletion and hypocalcemia or vitamin D depletion supplemented with calcium alone for 3, 7, or 14 days, vitamin D3 (6.5 nmol/day × 7 days), or 1,25(OH)2D3 (28 pmol/day × 7 days). Serum calcium was 1.28 ± 0.04 mM in hypocalcemic vitamin D-depleted rats, 1.47 ± 0.06 (NS), 1.8 ± 0.2 (P P < 0.0001) mM after 3, 7, or 14 days, respectively, of calcium supplementation; vitamin D3- or 1,25(OH)2D3- supplemented animals had serum calcium of 2.61 ± 0.04 or 2.48 ± 0.05 mM (P 67 < 0.0001 vs. hypocalcemic vitamin D-depleted rats). Rats with hypocalcemia and vitamin D depletion had significantly higher glucose concentrations (P < 0.0005) and lower insulin response during GTT than all other groups (P < 0.001). Differences in insulin sensitivity could not account for differences in response because exogenous insulin administration led to a similar drop in glucose concentrations in all groups, with the nadir averaging 51.7 ± 2.6% of initial values. To distinguish between calcium and the vitamin D system in the GTT response, rats were treated with a nonhypercalcemic analogue of 1,25(OH)2D3, OCT (28 pmol/day × 4-7days) with or without dietary calcium. Serum calcium was 1.23 ± 0.04 vs. 2.09 ± 0.02 mM in the absence or presence of dietary calcium (P < 0.0001), but normalization of GTT only happened in the presence of calcium. A time course of the calcium effect in vitamin D-depleted rats indicated that 7 days of high dietary calcium intake was needed to normalize GTT, with a significant correlation coefficient being observed between serum calcium and the maximum insulin response (r = 0.5172, P < 0.004). Our data indicate that vitamin D depletion with hypocalcemia is associated with normal insulin sensitivity but glucose intolerance caused by inadequate insulin secretion in response to glucose. Calcium alone contributes in normalizing GTT, whereas the 1,25(OH)2D3 analogue OCT is unable to normalize GTT at a dose equimolar to that of 1,25(OH)2D3 in the absence of calcium, suggesting that hypocalcemia predominates over vitamin D depletion in the glucose intolerance of vitamin D deficiency.