Omeprazole (OPZ) is a proton pump inhibitor commonly used for the treatment of gastric acid hypersecretion. Studies have revealed that use of OPZ can induce hepatotoxicity, but the mechanisms by which it induces liver injury are unclear. This study aimed to identify reactive metabolites of OPZ, determine the pathways of the metabolic activation, and define the correlation of the bioactivation with OPZ cytotoxicity. Quinone imine-derived glutathione (GSH), N-acetylcysteine (NAC), and cysteine (Cys) conjugates were detected in OPZ-fortified rat and human liver microsomal incubations captured with GSH, NAC, or Cys. The same GSH conjugates were detected in bile of rats and cultured liver primary cells after exposure to OPZ. Similarly, the same NAC conjugates were detected in urine of OPZ-treated rats. The resulting quinone imine was found to react with Cys residues of hepatic protein. CYP3A4 dominated the metabolic activation of OPZ. Exposure to OPZ resulted in decreased cell survival in cultured primary hepatocytes. Pretreatment with ketoconazole attenuated the susceptibility of hepatocytes to the cytotoxicity of OPZ.