Vitamin D deficiency is prevalent especially in men. Nevertheless, whether vitamin D deficiency impairs male reproduction remains under debate. The aim of this study is to investigate whether vitamin D deficiency has an impact on testicular development and spermatogenesis in mice. In the control group and vitamin D deficient (VDD) diet group, dams and their pups were fed with standard-chow diet and VDD diet, respectively. Interestingly, testicular weight and sperm quality are reduced, testicular germ cell proliferation is suppressed, and the percentage of mature seminiferous tubules is decreased in VDD diet-fed mice. Moreover, testicular testosterone (T) synthesis enzymes are down-regulated in VDD diet-fed mice. Correspondingly, serum and testicular T levels are reduced in VDD diet-fed mice. Importantly, fertility index is reduced and live fetuses are decreased when both males and females are fed with VDD diet. These results provide evidence that vitamin D deficiency impairs testicular development and spermatogenesis.