Background Drug interaction is one factor which may influence high-dose methotrexate (MTX) elimination. Proton pump inhibitors are commonly used as an adjuvant drugs in chemotherapy. However, the effect of proton pump inhibitors on high-dose MTX elimination is currently controversial. Objective To perform a systematic review and meta-analysis to assess the association between co-administration of proton pump inhibitors with plasma MTX concentration and delayed MTX elimination. Setting The Hospital of Kunming Medical University, China. Method We followed the PRISMA guidelines in this meta-analysis and systemic review. We searched PubMed, the Cochrane Database, Embase, the WHO International Clinical Trials Registry Platform, the Wanfang database, the Chinese National Knowledge Infrastructure, the VIP database and the Chinese BioMedical Literature Database. Main outcome measure The main outcome measures are: (1) the plasma MTX concentration at 24 h, 48 h and 72 h.; (2) the frequency of patients with delayed MTX elimination. Results Ten retrospective cohort studies were included in the meta-analysis, with a total of 2760 cycles of high-dose MTX treatment. A meta-analysis revealed that compared to patients who did not receive proton pump inhibitors, patients who received proton pump inhibitors had a significantly higher plasma MTX concentration at 24 h (mean difference 2.71 mu M, 95% confidence interval 0.55 to 4.87; p = 0.01) and at 48 h (mean difference 0.14 mu M, 95% confidence interval 0.06 to 0.21; p < 0.01) after the MTX infusion. Furthermore, delayed MTX elimination was more frequent in patients that received PPIs (risk ratio 0.59, 95% confidence interval 0.41 to 0.84; p = 0.004). Conclusion This systematic review and meta-analysis reveals that the co-administration of proton pump inhibitors with methotrexate is associated with delayed high-dose MTX elimination. Proton pump inhibitors should be cautiously given when co-administered with high-dose MTX treatment.