Metabolism of 25-hydroxyvitamin D3 (25OHD3) plays a central role in regulating the biological effects of vitamin D in the body. Although cytochrome P450-dependent hydroxylation of 25OHD3 has been extensively investigated, limited information is available on the conjugation of 25OHD3. In the present study, we report that 25OHD3 is selectively conjugated to 25OHD3-3-O-sulfate by human sulfotransferase 2A1 (SULT2A1) and that the liver is a primary site of metabolite formation. At a low (50 nM) concentration of 25OHD3, 25OHD3-3-O-sulfate was the most abundant metabolite, with an intrinsic clearance approximately 8-fold higher than the next most efficient metabolic route. In addition, 25OHD3 sulfonation was not inducible by the potent hPXR agonist, rifampicin. The 25OHD3 sulfonation rates in a bank of 258 different human liver cytosols were highly variable, but correlated with the rates of dehydroepiandrosterone sulfonation. Further analysis revealed a significant association between a common SNV within intron-1 of SULT2A1 (rs296361; MAF = 15% in Whites) and liver cytosolic SULT2A1 content as well as 25OHD3-3-O-sulfate formation rate, suggesting that variation in the SULT2A1 gene contributes importantly to inter-individual differences in vitamin D homeostasis. Finally, 25OHD3-3-O-sulfate exhibited high affinity for the vitamin D binding protein and was detectable in human plasma and bile, but not in urine samples. Thus, circulating concentrations of 25OHD3-3-O-sulfate appear to be protected from rapid renal elimination, raising the possibility that the sulfate metabolite may serve as a reservoir of 25OHD3 in vivo, and contribute indirectly to the biological effects of vitamin D.