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Dietary supplements containing usnic acid have been increasingly marketed for weight loss over the past decades, even though incidences of severe hepatotoxicity and acute liver failure due to their overuse have been reported. To date, the toxic mechanism of usnic acid-induced liver injury at the molecular level still remains to be fully elucidated. Here, we conducted a transcriptomic study on usnic acid using a novel in vitro hepatotoxicity model employing human induced pluripotent stem cell (iPSC)-derived hepatocytes. Treatment with 20 μM usnic acid for 24 h caused 4272 differentially expressed genes (DEGs) in the cells. Ingenuity Pathway Analysis (IPA) based on the DEGs and gene set enrichment analysis (GSEA) using the whole transcriptome expression data concordantly revealed several signaling pathways and biological processes that, when taken together, suggest that usnic acid caused oxidative stress and DNA damage in the cells, which further led to cell cycle arrest and eventually resulted in cell death through apoptosis. These transcriptomic findings were subsequently corroborated by a variety of cellular assays, including reactive oxygen species (ROS) generation and glutathione (GSH) depletion, DNA damage (pH2AX detection and 8-hydroxy-2'-deoxyguanosine [8-OH-dg] assay), cell cycle analysis, and caspase 3/7 activity. Collectively, the results of the current study accord with previous in vivo and in vitro findings, provide further evidence that oxidative stress-caused DNA damage contributes to usnic acid-induced hepatotoxicity, shed new light on molecular mechanisms of usnic acid-induced hepatotoxicity, and demonstrate the usefulness of iPSC-derived hepatocytes as an in vitro model for hepatotoxicity testing and prediction., (© 2024 The Authors. Journal of Applied Toxicology published by John Wiley & Sons Ltd.)