Although vitamin D (vD) deficiency is common in breastfed infants and their mothers during pregnancy and lactation, a standardized global comparison is lacking. We studied the prevalence and risk factors for vD deficiency using a standardized protocol in a cohort of breastfeeding mother-infant pairs, enrolled in the Global Exploration of Human Milk Study, designed to examine longitudinally the effect of environment, diet and culture. Mothers planned to provide breast milk for at least three months post-partum and were enrolled at four weeks postpartum in Shanghai, China (n=112), Cincinnati, Ohio (n=119), and Mexico City, Mexico (n=113). Maternal serum 25(OH)D was measured by radioimmunoassay (<50 nmol/L was categorized as deficient). Serum 25(OH)D was measured in a subset of infants (35 Shanghai, 47 Cincinnati and 45 Mexico City) seen at 26 weeks of age during fall and winter seasons. Data collected prospectively included vD supplementation, season and sun index (sun exposure×body surface area exposed while outdoors). Differences and factors associated with vD deficiency were evaluated using appropriate statistical analysis. vD deficiency in order of magnitude was identified in 62%, 52% and 17% of Mexican, Shanghai and Cincinnati mothers, respectively (p<0.001). In regression analysis, vD supplementation (p<0.01), obesity (p=0.03), season (p=0.001) and sites (p<0.001) predicted maternal vD status. vD deficiency in order of magnitude was found in 62%, 28%, and 6% of Mexican, Cincinnati and Shanghai infants, respectively (p<0.001). Season (p=0.022), adding formula feeding (p<0.001) and a higher sun index (p=0.085) predicted higher infant vD status. vD deficiency appears to be a global problem in mothers and infants, though the prevalence in diverse populations may depend upon sun exposure behaviors and vD supplementation. Greater attention to maternal and infant vD status starting during pregnancy is warranted worldwide.