Pexidartinib, a tyrosine kinase inhibitor, was approved by the U.S. Food and Drug Administration in 2019 for treating adult patients with symptomatic tenosynovial giant cell tumors. Because of its hepatotoxicity risks, pexidartinib received a boxed warning; however, mechanistic studies on this hepatotoxicity remain limited. In this study, we demonstrate that pexidartinib decreases cell viability in primary human hepatocytes and hepatic HepG2 cells. A 24-h treatment with pexidartinib led to apoptosis in HepG2 cells, as evidenced by increased caspase 3/7 activity and the induction of cleaved PARP and γ-H2A.X. Pexidartinib-induced endoplasmic reticulum (ER) stress was observed at early time points of 2 h and 5 h. The ER stress inhibitor 4-phenylbutyrate (4-PBA) partially attenuated pexidartinib-induced cytotoxicity. Additionally, mitochondrial dysfunction caused by pexidartinib was indicated by a strong inhibition of mitochondrial respiratory complexes II, IV, and V, a loss of mitochondrial potential, and greater toxicity in galactose media compared to glucose media. Pexidartinib also induced autophagy, as demonstrated by the formation of autophagosomes and autophagic flux. We investigated the role of cytochrome P450 (CYP)-mediated metabolism in pexidartinib-induced cytotoxicity by using HepG2 cell lines engineered to express 14 individual CYPs. The results indicated that CYP1A1, 2C9, 3A4, and 3A5 were involved in pexidartinib metabolism. Inhibition of CYP1A1, 2C9, and 3A4/5 significantly increased pexidartinib-induced cytotoxicity in primary human hepatocytes. Collectively, these data suggest that pexidartinib induced apoptosis, ER stress, mitochondria dysfunction, and autophagy in hepatic cells, and CYPs-mediated metabolism played a protective role in reducing pexidartinib toxicity.