Drug‐induced liver injury (DILI) is a serious and frequently occurring issue in drug development. The c‐Jun N‐terminal kinase (JNK) signaling pathway plays an important role in many diseases; hepatocyte nuclear factor‐1α (HNF‐1α) and glutathione S‐transferase A1 (GSTA1) are important in regulating liver‐specific genes expressions and affecting drug metabolism. Oltipraz is used to treat liver cirrhosis by improving liver function, and C2‐ceramide is a pro‐apoptotic lipid that regulates multiple signaling pathways. In this study, we investigated the function of the JNK signaling pathway with HNF‐1α and GSTA1 in a cellular model of DILI and whether oltipraz and C2‐ceramide exert effects via the JNK pathway. The results showed that inhibiting JNK could ameliorate APAP‐induced hepatocyte injury, reduced oxidative stress, suppressed JNK and c‐Jun activation, and hepatocyte apoptosis. Meanwhile, the mRNA and protein expressions of HNF‐1α and GSTA1 were increased significantly compared to control conditions. The effect of oltipraz (8 μmol/L) was similar to a JNK inhibitor and significantly increased HNF‐1α/GSTA1 expression, but oltipraz combined with JNK inhibitor did not show a synergistic effect. Although C2‐ceramide (8 μmol/L) aggravated hepatocyte injury and apoptosis, exacerbated oxidative stress, increased phosphorylation of JNK and c‐Jun, and markedly decreased HNF‐1α/GSTA1 expression, C2‐ceramide combined with JNK inhibitor could partially alleviate these alterations. These results demonstrated that the JNK signaling pathway with HNF‐1α/GSTA1 are involved in the process of DILI. Inhibiting JNK up‐regulated HNF‐1α and GSTA1 expressions which could attenuate hepatocyte injury. Oltipraz and C2‐ceramide might affect the expression of HNF‐1α/GSTA1 though JNK signaling.