The mechanism(s) by which vitamin D₃ regulates female reproduction is minimally understood. We tested the hypothesis that peripubertal vitamin D₃ deficiency disrupts hypothalamic-pituitary-ovarian physiology. To test this hypothesis, we used wild-type mice and Cyp27b1 (the rate-limiting enzyme in the synthesis of 1,25-dihydroxyvitamin D₃) null mice to study the effect of vitamin D₃ deficiency on puberty and reproductive physiology. At the time of weaning, mice were randomized to a vitamin D₃-replete or -deficient diet supplemented with calcium. We assessed the age of vaginal opening and first estrus (puberty markers), gonadotropin levels, ovarian histology, ovarian responsiveness to exogenous gonadotropins, and estrous cyclicity. Peripubertal vitamin D₃ deficiency significantly delayed vaginal opening without affecting the number of GnRH-immunopositive neurons or estradiol-negative feedback on gonadotropin levels during diestrus. Young adult females maintained on a vitamin D₃-deficient diet after puberty had arrested follicular development and prolonged estrous cycles characterized by extended periods of diestrus. Ovaries of vitamin D₃-deficient Cyp27b1 null mice responded to exogenous gonadotropins and deposited significantly more oocytes into the oviducts than mice maintained on a vitamin D₃-replete diet. Estrous cycles were restored when vitamin D₃-deficient Cyp27b1 null young adult females were transferred to a vitamin D₃-replete diet. This study is the first to demonstrate that peripubertal vitamin D₃ sufficiency is important for an appropriately timed pubertal transition and maintenance of normal female reproductive physiology. These data suggest vitamin D₃ is a key regulator of neuroendocrine and ovarian physiology.