〔Abstract〕 Objective To study the effect of non-small cell lung cancer(A549 cells) on the sensitivity of the chemotherapy drug pemetrexed(Pe) under hypoxia environment. Methods The A549 cell line was cultured in groups and divided into cell control group, normoxia solvent group, normoxia dosing group, hypoxia cell group, hypoxia solvent group and hypoxia dosing group. First, MTT was used to detect the effect of Pe on the proliferation of A549 cells, thereby screening out the suitable experimental concentration of Pe. Then Hoechst staining was used to observe the apoptosis of these six cell groups. Later, Western blot was applied to detect the expressions of apoptosis-related proteins Bcl-2, Bax, and p53. Results MTT results showed that compared with the normoxia control group, the cell inhibition rate after Pe treatment gradually increased(P<0.05), and the inhibition rate of A549 cells was directly proportional to the action time and concentration of Pe. The results of Hoechst staining indicated that cell apoptosis rate increased in the normoxia-dosed group compared with the normoxia control group(P<0.05). But compared with normoxia-dosed group, the cell apoptosis rate decreased under hypoxic environment(P<0.05). Western Blot experiment showed an increase in p53 protein and a decrease in Bcl-2 protein expression in the normoxia dosing group compared to the normoxia control group(P<0.05). There was no change of p53 protein expression identified in the hypoxia dosing group, but Bcl-2 protein expression decreased(P<0.05); compared with the hypoxic cell group, Bcl-2 protein expression decreased(P<0.05) in the hypoxia dosed group, while p53 protein expression was unchanged. Conclusion Hypoxia environment reduces the apoptotic effect of Pe on non-small cell lung cancer A549 cells, and thus reducing the sensitivity of A549 cells to pemetrexed.