Ochratoxin A (OTA) widely exists in raw food and feed materials and can induce liver damage and toxicity. However, the mechanisms of OTA-induced hepatotoxicity were largely unknown. Thus, our study aimed to uncover the vital genes relevant to OTA-induced hepatotoxicity in broiler chickens. Gene expression data of chicken embryo primary hepatocytes (CEPHs) in OTA-treated and control groups were obtained from the GEO database. Totally 1407 differentially expressed genes (DEGs) were selected, of which 850 and 557 genes were up- and downregulated in OTA-treated CEPHs. Gene ontology (GO) enrichment revealed that the DEGs were in connection with various biological processes, such as signal transduction, extracellular matrix organization, axon guidance, cell division, cholesterol homeostasis, proteolysis, microtubule cytoskeleton organization, and chromosome segregation. Pathway enrichment showed that the DEGs were related to metabolic pathways, ferroptosis, calcium, FoxO, Wnt, cell cycle, apoptosis, calcium, and cell adhesion molecules signaling pathways. Furthermore, the hub genes, including CDK1, DLGAP5, KIF2C, VCL, ITGB3, and ZYX, were identified as hub genes potentially contributing to OTA-induced hepatotoxicity. Taken together, this study provides valuable insights into the mechanisms underlying OTA-induced hepatotoxicity in broiler chickens.