Drug‑induced liver injury has become a serious public health problem. Although the mechanism of acetaminophen (APAP)‑induced liver injury has been studied for decades it has not been fully elucidated. In‑depth study into the mechanisms underlying APAP‑induced liver injury may provide useful information for more effective prevention and treatment. In the present study, the role of C‑X‑C motif chemokine ligand‑9 (CXCL9) in APAP‑induced liver injury was investigated thus providing a novel direction for the prevention and treatment of drug hepatitis. A total of 20 fasting male patients ingested APAP tablets at Nanjing First Hospital. In addition, wild type (WT) mice were treated with 250 mg/kg APAP or isodose PBS for 1, 3, 6 and 12 h, respectively. Results from reverse‑transcription‑quantitative polymerase chain reaction analyses demonstrated that CXCL9 mRNA levels were increased in the blood of patients who took APAP in a fasting state and in the livers of APAP‑treated WT mice, compared with their respective controls. Hepatocyte apoptosis in the liver tissue of APAP‑treated mice decreased following administration of a CXCL9 neutralizing antibody. Caspase‑3, caspase‑8 and phosphorylated‑AKT (S437) were activated in primary hepatocytes isolated from WT mice following CXCL9 treatment. However, no significant differences in expression of caspase‑3, caspase‑8 and p‑AKT (S437) were detected in hepatocytes isolated from C‑X‑C motif chemokine receptor 3 (CXCR3)‑/‑mice following CXCL9 treatment. After CXCL9 administration, WT mice exhibited higher serum levels of aspartate transaminase and increased caspase‑3 and caspase‑8 activity in liver tissue compared with controls. The same trends were not observed in CXCR3‑/‑mice. In conclusion, CXCL9 regulated APAP‑induced liver injury through stimulation of hepatocyte apoptosis via binding to CXCR3. These findings provide a novel prevention and treatment strategy for DILI.