Sulfated vitamin D metabolites are gaining attention in maternal health and infant nutrition. In pregnant women, serum concentrations of 25-hydroxyvitamin D3-sulfate (25OHD-S) are similar to those of 25-hydroxyvitamin D3 (25OHD). For newborn infants, concentrations of 25OHD-S in serum are greater than 25OHD. Vitamin D3-sulfate (VitD-S) in breastmilk has been suggested in the past, yet its presence and function remain unclear. We hypothesized that VitD-S and 25OHD-S are significant components of human milk and contribute to infant nutrition via properties and mechanisms uncommon to vitamin D3 (VitD). To evaluate VitD-S and 25OHD-S in milk, we first developed analytical methods by LC-MS/MS and noted two key features that affect nutrient bioavailability: instability and solubility. Next, a cohort of 20 healthy lactating mothers were recruited to measure VitD-S and 25OHD-S, in milk and serum, before (VitD ≤600 IU/day) and after 28 days of high-dose supplementation (VitD 5,000 IU/day). At baseline, mothers were vitamin D-sufficient (mean±SD serum 25OHD 69±19 nmol/L) and had significantly greater 25OHD-S (91±19 nmol/L, p <0.001). Baseline milk contained both VitD-S (6.4±3.9 nmol/L) and 25OHD-S (0.47±0.09 nmol/L). After high-dose supplementation, serum VitD and 25OHD in the mothers increased, but concentrations of 25OHD-S or VitD-S, in serum and milk, remained unaffected. Correlational assessments identified potential features of the sulfation pathways including an observation that VitD-S in milk was distinct and unrelated to precursors in serum. Last, we collected fecal samples from breastfeeding infants (ages 1-6 months) to evaluate the potential impact of gut microbiota on the digestion of the sulfated metabolites. Sulfatase activities of fecal collections from exclusively breastfed infants (n =11) were greater than the activities from infants who consume formula in addition to breastmilk (n =4, p =0.024). Sulfatase activity, independent of diet, was associated with bacterial abundance of the genus Klebsiella in the infant microbiome (n=8). Taken together, our studies highlight important considerations of sulfated vitamin D metabolites including enhanced solubility, physiological regulation, degradation during milk storage, and hydrolysis by the gut bacteria. Incorporating these factors into future s