Scopoleitin (SP), a bioactive compound from many edible plants and fruits, exerts a wide range of biological activities, however the role and mechanism of SP in acetaminophen (APAP)-induced hepatotoxicity remains unclear. In this study, we verified the protective effect of SP on APAP-induced liver injury (AILI) hepatotoxicity and explore the underlying molecular mechanisms. Here, we showed that SP alleviated AILI by reducing serum alanine transaminase (ALT) and aspartate aminotransferase (AST) levels, hepatic histopathological damage, inflammation, and liver cell apoptosis. In addition, SP attenuated the accumulation of malondialdehyde (MDA) and exhaustion of glutathione (GSH) levels and increased the superoxide dismutase (SOD) levels induced by APAP. Consistently, SP significantly reduced the gene transcription of cytochrome P450 (CYP)2E1, CYP1A2, and CYP3A11 in the livers of mice induced by APAP. Moreover, SP pretreatment effectively promoted the expression of Nrf2, Keap1, and its signal downstream HO-1, NQO1, GCLc, and GCLm, suggesting the activation of the Nrf2 signaling pathway. SP inhibited APAP-induced hepatocyte apoptosis by regulating the protein levels of apoptosis-related proteins (cytochrome C, Bax, Caspase-3, Bcl2, and PARP). SP suppressed APAP-induced expression of NLRP3 and reduced the levels of proinflammatory factors, including tumor necrosis factor-alpha (TNF-α), F4/80, Caspase-1, and interleukin (IL)-1 beta (IL-1β). Moreover, SP downregulated APAP-induced high-mobility group box 1 (HMGB1) and toll-like receptor 4 (TLR4) expression, inhibited nuclear factor kappa-B (NF-κB) and MAPK activation. Taken together, our study reveals the protective roles of SP against AILI through the downregulation of NLRP3 expression, and the inhibition of the Nrf2/HMGB1/TLR4/NF-κB signaling pathways.