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It was 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. Proteomic analysis of HCAECs exposed to APAP showed upregulation of CYP20A1, together with proteins involved in the pentose phosphate pathway and maintaining redox homeostasis. Proteomic analyses of mesenteric arteries from rats administered intravenous APAP are consistent with a key role of the vascular wall in APAP metabolism, with similar proteomic pathway changes identified in HCAECs. These changes occurred over a short timeframe and were not seen in the corresponding proteomic analyses of liver tissue. Intracellular thiols were depleted in HCAECs upon APAP treatment, which was partially attenuated by ketoconazole, consistent with the involvement of cytochrome P450 enzymes 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 the absence of chloride ions, consistent 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.