Viral infections are often linked to altered drug metabolism in patients; however, the underlying molecular mechanisms remain unclear. Here we describe a mechanism by which activation of antiviral responses by the synthetic double-stranded RNA ligand, polyinosinic-polycytidylic acid (polyI:C), leads to decreased acetaminophen (APAP) metabolism and hepatotoxicity. PolyI:C administration down-regulates expression of retinoic X receptor-α (RXRα) as well as its heterodimeric partner pregnane X receptor (PXR) in mice. This down-regulation results in suppression of downstream cytochrome P450 enzymes involved in conversion of APAP to its toxic metabolite. Although the effects of polyI:C on drug metabolism are often attributed to interferon production, we report that polyI:C can decrease APAP metabolism in the absence of the type I interferon receptor. Furthermore, we demonstrate that polyI:C can attenuate APAP metabolism through both its membrane-bound receptor, Toll-like receptor 3 (TLR3), as well as cytoplasmic receptors. Conclusion: This is the first study to illustrate that in vivo administration of polyI:C affects drug metabolism independent of type I interferon production or in the absence of TLR3 through crosstalk between nuclear receptors and antiviral responses. (HEPATOLOGY 2011;)