Vitamin D deficiency reduces mating success and fertility in female rats, but it is not known if the reduction in reproductive performance is a direct action of vitamin D or the hypocalcemia associated with vitamin D deficiency. The effect of vitamin D deficiency with normocalcemia on fertility and reproductive capacity in female rats was investigated. Female weanling rats were maintained on vitamin D-deficient or vitamin D-replete diets until maturity and mated to age-matched, normal, vitamin D-replete males. Three groups of vitamin D-deficient females were maintained on diets varying in calcium and Pi concentrations to test the effect of vitamin D deficiency with different serum calcium and Pi concentrations on reproductive performance. Vitamin D-deficient females were capable of reproduction, but successful matings by all groups of vitamin D-deficient females were markedly reduced regardless of serum calcium concentration, when compared with matings with vitamin D-replete females. Fertility was also drastically reduced in litters from all groups of vitamin D-deficient females regardless of serum calcium concentration, when compared with litters from vitamin D-replete females. Vitamin D-deficient female rats that received vitamin D or 1,25-dihydroxyvitamin D3 were capable of successfully mating and giving rise to normal, healthy litters. These results indicate that vitamin D and not hypocalcemia is directly responsible for reduced reproductive capacity and fertility in vitamin D-deficient female rats.