Abstract: Objective To investigate the anti-proliferative and apoptotic effect of different sequences of combined pemetrexed and gefitinib on human lung adenocarcinoma cells A549 and PC-9, and to probe the possible mechanisms. Methods MTT assay was used to measure cell proliferation. Flow cytometry using annexin V-FITC/PI staining was employed to measure cell apoptosis and cell cycle. The expression of phosphorylation of EGFR, AKT, ERK and TS protein were detected by Western blot. Results Sequential pemetrexed followed by gefitinib or cocurrent pemetrexed and gefitinib remarkably enhanced the antiproliferation effects (P<0.05). Sequential pemetrexed followed by gefitinib or cocurrent pemetrexed and gefitinib enhanced the apoptosis effect on PC-9 and A549 cells. Pemetrexed induced the phosphorylation of EGFR, AKT and ERK, while gefitinib significantly suppressed the phosphorylation of EGFR,AKT,ERK and TS expression. Gefitinib mainly blocked the cells in G0/G1 phase and pemetrexed blocked the cells in S phase. The percentage of cells in G2/M phase was increased in sequential pememtrexed followed by gefitinib or cocurrent pemetexed and gefitinib group (P<0.05). Sequential pemetrexed followed by gefitinib or concurrent pemetrexed and gefitinib had a more significant effect on decreasing the expression of p-EGFR，p-AKT and p-ERK. Conclusion Both concurrent pemetrexed and gefitinib or sequential pemetrexed followed by gefitinib have synergistic effects on the expression of p-EGFR,p-AKT and p-ERK, moreover sequential pemetrexed followed by gefitinib has more synergistic effects than concurrent pemetrexed and gefitinib. The phosphorylation of EGFR,AKT and ERK induced by pemetrexed and TS expression suppressed by gefitinib may contribute to the synergistic effects.