Drug-induced liver injury (DILI) is a major cause of acute liver failure and drug withdrawal. Cytochrome P450 (CYP) 2E1 is involved in the metabolism of several drugs, and can induce liver injury through the production of toxic metabolites and the generation of reactive oxygen species. This study aimed to elucidate the role of Wnt/ß-catenin signaling in CYP2E1 regulation for drug-induced hepatotoxicity. To achieve this, mice were administered cisplatin or acetaminophen (APAP) 1 h after treatment with the CYP2E1 inhibitor dimethyl sulfoxide (DMSO), and histopathological and serum biochemical analyses were performed. APAP treatment induced hepatotoxicity, as evidenced by an increase in liver weight and serum ALT levels. Moreover, histological analysis indicated severe injury, including apoptosis, in the liver tissue of APAP-treated mice, which was confirmed by TUNEL assay. Additionally, APAP treatment suppressed the antioxidant capacity of the mice and increased the expression of the DNA damage markers γ-H2AX and p53. However, these effects of APAP on hepatotoxicity were significantly attenuated by DMSO treatment. Furthermore, the activation of Wnt/ß-catenin signaling using the Wnt agonist CHIR99021 (CHIR) increased CYP2E1 expression in rat liver epithelial cells (WB-F344), whereas treatment with the Wnt/ß-catenin antagonist IWP-2 inhibited nuclear ß-catenin and CYP2E1 expression. Interestingly, APAP-induced cytotoxicity in WB-F344 cells was exacerbated by CHIR treatment and suppressed by IWP-2 treatment. Overall, these results showed that the Wnt/ß-catenin signaling is involved in DILI through the upregulation of CYP2E1 expression by directly binding the transcription factor ß-cat/TCF to the Cyp2e1 promoter, thus exacerbating DILI. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-023-00180-6.