High-dose methotrexate (HD-MTX) is widely used to treat osteosarcoma. However, some patients develop hepatic toxicity, leading to dose modification and delays in the scheduled chemotherapy. The present study aimed to identify the risk factors of hepatotoxicity in patients with osteosarcoma.We conducted a retrospective study of patients with osteosarcoma treated with HD-MTX between January 2014 and June 2020 at the National Cancer Center Hospital, Japan. The risk factors for MTX-induced hepatotoxicity (≥grade 3) were identified using multivariate logistic regression analysis.The final analysis included 88 courses of 36 patients. Hepatotoxicity occurred in 51 (58.0%) of the 88 courses. Female sex, MTX dose (>10.2 g/m), and serum calcium concentration (>9.3 mg/dl) were identified as risk factors for HD-MTX-induced hepatotoxicity.Identifying the risk factors of HD-MTX-induced hepatotoxicity may contribute to improvements in the safety and management of HD-MTX therapy.