〔Abstract〕 Objective To investigate the effect of hypoxia on pemetrexed (PE)-mediated apoptosis in non-small cell lung cancer cells. Methods A549 cells were cultured under 21% O₂ (normoxia) or 1% O₂ (hypoxia), and treated with 8 μmol/L PE for 48 h. The cells were divided into normoxic control group, normoxic vehicle group, normoxic drug-treated group, hypoxic control group, hypoxic vehicle group and hypoxic drug-treated group. Trypan blue staining was used to observe cell death after PE intervention under normoxia and hypoxia. Flow cytometry was adopted to detect cell apoptosis rate. Wound healing assay was used to assess cell migration. Western blot was performed to detect the protein levels of MRP-1, Cleaved Caspase-3, E-Cadherin and N-Cadherin. Results Trypan blue staining results showed that the cell death rate was reduced in PE-treated cells under hypoxia compared with the normoxic PE group (P<0.001). Flow cytometry results indicated that PE induced cell apoptosis under both normoxic and hypoxic conditions.However, the apoptosis rate of the normoxic PE group was significantly higher than that of the hypoxic PE group (P<0.000 1). Wound healing assay showed that the migration distance of A549 cells decreased after PE treatment for 24 h and 48 h under normoxia and hypoxia when compared with the control group (P<0.05), while no significant difference was found between the normoxic PE group and the hypoxic PE group. Western blot results revealed that MRP-1 expression was markedly higher in hypoxic PE-treated cells than in normoxic PE-treated cells (P<0.000 1). Compared with the control group, Cleaved Caspase-3 expression was upregulated in A549 cells following PE intervention under normoxia (P<0.05), whereas no obvious alteration in Cleaved Caspase-3 expression was detected in hypoxic PE-treated cells. Under both normoxic and hypoxic conditions, PE treatment downregulated the expression of E-Cadherin (P<0.000 1 ，P<0.001) and N-Cadherin (P<0.05 ，P<0.000 1). In addition, the expression levels of E-Cadherin and N-Cadherin in the hypoxic PE group were significantly lower than those in the normoxic PE group (P<0.000 1). Conclusion Hypoxia significantly attenuates the pro-apoptotic effect of PE on A549 cells and blocks Cleaved Caspase-3 activation. This mechanism may be related to hypoxia-induced MRP-1 overexpression and reduced drug accumulation. Interfering with MRP-1 or restoring the activity of the apoptosis pathway may serve as a novel strategy to overcome hypoxia-induced drug resistance.