Abstract: Objective To investigate the influence of drug combinations on high-dose methotrexate(MTX)- induced nephrotoxicity and MTX blood concentrations. Methods We retrospectively analyzed the clinical data of 178 childhood acute lymphoblastic leukemia(ALL) patients who received 633 courses of high-dose MTX therapies in the First Affiliated Hospital of Guangxi Medical University from Sep. 2009 to Sep. 2014. The patients were divided into two groups based on the medications taken: control group was treated with routine high-dose MTX, and the drug combination group was additionally given penicillins, non-steroidal anti-inflammatory drugs or proton pump inhibitors on the basis of control group. The incidences of acute kidney injury(AKI) and MTX blood concentrations were compared between two groups. Results The combinations of piperacillin, ibuprofen and omeprazole were observed in 84 courses of high-dose MTX therapies. The overall incidence of AKI in drug combination group was significantly higher than that in control group(P<0.05), which mainly manifested in a significantly increased incidences of moderate and severe AKI (P<0.05). MTX blood concentrations at each time point in drug combination group were significantly higher than those in control group(P<0.05). The days needed for MTX concentrations decreasing to the safe level in drug combination group were significantly longer than that in control group(P<0.05). Conclusion The combination of piperacillin, ibuprofen and omeprazole in high-dose MTX chemotherapy may lead to the excretion delay of MTX and increase the risk of renal toxicity. Such drug combinations should be avoided in the high-dose MTX therapies.