ARTICLE ABSTRACTVemurafenib is a revolutionary treatment for melanoma, but the magnitude of therapeutic response is highly variable, and the rapid acquisition of resistance is frequent. Here, we examine how vemurafenib disposition, particularly through cytochrome P450-mediated oxidation pathways, could potentially influence these outcomes using a panel of knockout and transgenic humanized mouse models. We identified CYP3A4 as the major enzyme involved in the metabolism of vemurafenib in in vitro assays with human liver microsomes. However, mice expressing human CYP3A4 did not process vemurafenib to a greater extent than CYP3A4-null animals, suggesting that other pregnane X receptor (PXR)–regulated pathways may contribute more significantly to vemurafenib metabolism in vivo. Activation of PXR, but not of the closely related constitutive androstane receptor, profoundly reduced circulating levels of vemurafenib in humanized mice. This effect was independent of CYP3A4 and was negated by cotreatment with the drug efflux transporter inhibitor elacridar. Finally, vemurafenib strongly induced PXR activity in vitro, but only weakly induced PXR in vivo. Taken together, our findings demonstrate that vemurafenib is unlikely to exhibit a clinically significant interaction with CYP3A4, but that modulation of bioavailability through PXR-mediated regulation of drug transporters (e.g., by other drugs) has the potential to markedly influence systemic exposure and thereby therapeutic outcomes. Cancer Res; 75(21); 4573–81. ©2015 AACR.