We have shown previously that a metabolite of acetaminophen (APAP), N-acetyl-p-benzoquinone imine (NAPQI), is a potent vasodilator, which could underlie the hypotension observed when APAP is administered intravenously. However, it is unknown whether APAP metabolism to NAPQI is possible in the vasculature. In this study, we examine the hypothesis that APAP is metabolized by cytochrome P450 enzymes within the endothelium, which may be accelerated in critically ill patients by the presence of elevated myeloperoxidase (MPO). Exposure of human coronary artery endothelial cells (HCAECs) to APAP resulted in the formation of protein-bound APAP adducts, together with a loss of metabolic activity. Proteomic analysis of HCAECs exposed to APAP showed upregulation of CYP20A1 and cytochrome P450 reductase (POR), together with proteins involved in the pentose phosphate pathway and maintaining redox homeostasis. Furthermore, proteomic analysis of mesenteric arteries and livers from rats administered intravenous APAP showed that APAP metabolism likely took place in the vascular wall and not in the liver. Moreover, similar proteomic pathway changes were identified in HCAECs and mesenteric arteries. Intracellular thiols were depleted in HCAECs upon APAP treatment, which was partially attenuated by ketoconazole, consistent with the involvement of cytochrome P450 enzymes (CYP) in the metabolism of APAP to a thiol-reactive metabolite such as NAPQI. Evidence was also obtained for the metabolism of APAP to a thiol-reactive intermediate by MPO, in accordance with NAPQI formation. Taken together, these data provide a putative mechanism to explain the presentation of hypotension in critically ill patients following IV APAP administration.