Diclofenac (DCLF) is a nonsteroidal anti-inflammatory drug that is associated with idiosyncratic adverse drug reactions in humans. Previous studies revealed a crucial role for intestine-derived bacteria and/or lipopolysaccharide (LPS) in DCLF-induced hepatotoxicity. We further explored this mechanism by conducting gene expression analysis of livers from rats treated with a hepatotoxic dose of DCLF (100 mg/kg) with or without oral antibiotic pretreatment. Genes for which expression was altered by DCLF were divided into two groups: genes with expression altered by antibiotic treatment and those unaffected by antibiotics. The former group of genes represented the ones for which DCLF-induced alterations in expression depended on intestinal bacteria. The expression of the latter group of genes was probably changed by direct effect of DCLF rather than by intestinal bacteria. Functional analysis of genes in the former group revealed LPS-related signaling, further suggesting a role for bacterial endotoxin in the liver injury. Functional analysis of genes in the latter group revealed changes in signaling pathways related to inflammation, hypoxia, oxidative stress, the aryl hydrocarbon receptor, and peroxisome proliferator-activated receptor alpha. Neutrophil depletion failed to protect from DCLF-induced hepatotoxicity, suggesting that intestinal bacteria contribute to liver injury in a neutrophil-independent manner. Hypoxia occurred in the livers of rats treated with DCLF, and hypoxia in vitro rendered hepatocytes sensitive to DCLF-induced cytotoxicity. These results support the hypothesis that intestinal bacteria are required for DCLF-induced hepatotoxicity and suggest that hypoxia plays an important role in the pathogenesis.