Abstract An acetaminophen (APAP) overdose is the most common cause of acute liver failure in the United States. A hallmark characteristic of APAP hepatotoxicity is centrilobular necrosis. General, innate mechanisms such as lower amounts of GSH and higher cytochrome P450 2e1 expression in pericentral (PC) hepatocytes are known to contribute to the differences in susceptibility to cell injury between periportal (PP) hepatocytes and PC hepatocytes. Although a sequence of molecular events involving formation of the reactive metabolite N-acetyl-p-benzoquinone imine, GSH depletion, oxidative stress, and c-Jun N-terminal kinase activation define the early cell stress trajectory following APAP exposure, their activation in PC versus PP hepatocytes is not well characterized. By using single-cell RNA-sequencing, we provide the first reconstruction of the early transcriptomic APAP liver lobule after validation of our methodology using human liver single-cell RNA-sequencing data. Two hours after APAP treatment, we find that PP hepatocytes progress along the APAP stress axis to oxidative stress, before resolving injury due to innate and adaptive mechanisms. However, PC hepatocytes continue along this stress axis as indicated by activation of mitogen-activated protein kinase genes, which is absent in PP hepatocytes. We also identify a population of glutamine synthetase enriched PC hepatocytes in close proximity to the central vein, where a stepwise induction of a stress program culminated in cell death. Collectively, these findings elucidate a molecular sequence of events distinguishing the differential response to APAP exposure between PP and PC hepatocytes and identify a subset of uniquely susceptible PC hepatocytes.