Epidemiologic studies have linked gestational Vitamin D deficiency to respiratory diseases, although mechanisms have not been defined. We hypothesized that antenatal Vitamin D deficiency would impair airway development and alveolarization in a mouse model. We studied the effect of antenatal Vitamin D deficiency by inducing it in pregnantmice and then compared lung development and function in their offspring to littermate controls. Postnatal Vitamin D deficiency and sufficiency models from each group were also studied.We developed a novel tracheal ultrasound imaging technique to measure tracheal diameter in vivo. Histological analysis estimated tracheal cartilage total area and thickness.Wefound that vitaminD-deficient pups had reduced tracheal diameter with decreased tracheal cartilage minimal width. Vitamin D deficiency increased airway resistance andreduced lung compliance, and led to alveolar simplification. Postnatal Vitamin D supplementation improved lung function and radial alveolar count, a parameter of alveolar development, but did not correct tracheal narrowing. We conclude that antenatal vitaminDdeficiency impairs airway andalveolar development and limits lung function. Reduced tracheal diameter, cartilage irregularity, and alveolar simplification in Vitamin D-deficient mice may contribute to increased airways resistance and diminished lung compliance. VitaminDsupplementation after birth improved lung function and, potentially, alveolar simplification, but did not improve defective tracheal structure. This mouse model offers insight into the mechanisms of Vitamin D deficiency-associated lung disease and provides an in vivo model for investigating preclinical preventive and therapeutic strategies.