Vitamin D₃ is critical for the regulation of calcium and phosphate homeostasis. In some individuals, mineral homeostasis can be disrupted by long-term therapy with certain antiepileptic drugs and the antimicrobial agent rifampin, resulting in drug-induced osteomalacia, which is attributed to vitamin D deficiency. We now report a novel CYP3A4-dependent pathway, the 4-hydroxylation of 25-hydroxyvitamin D₃ (25OHD₃), the induction of which may contribute to drug-induced vitamin D deficiency. The metabolism of 25OHD₃ was fully characterized in vitro. CYP3A4 was the predominant source of 25OHD₃ hydroxylation by human liver microsomes, with the formation of 4β,25-dihydroxyvitamin D₃ [4β,25(OH)₂D₃] dominating (V_max/K_m = 0.85 ml · min⁻¹ · nmol enzyme⁻¹). 4β,25(OH)₂D₃ was found in human plasma at concentrations comparable to that of 1α,25-dihydroxyvitamin D₃, and its formation rate in a panel of human liver microsomes was strongly correlated with CYP3A4 content and midazolam hydroxylation activity. Formation of 4β,25(OH)₂D₃ in primary human hepatocytes was induced by rifampin and inhibited by CYP3A4-specific inhibitors. Short-term treatment of healthy volunteers (n = 6) with rifampin selectively induced CYP3A4-dependent 4β,25(OH)₂D₃, but not CYP24A1-dependent 24R,25-dihydroxyvitamin D₃ formation, and altered systemic mineral homeostasis. Our results suggest that CYP3A4-dependent 25OHD₃ metabolism may play an important role in the regulation of vitamin D₃ in vivo and in the etiology of drug-induced osteomalacia.