Pemetrexed is an antifolate agent which has been used for treating malignant pleural mesothelioma and nonsmall lung cancer in the clinic as a chemotherapeutic agent. In this study, pemetrexed inhibited cell growth andinduced G1 phase arrest in the A549 cell line. To explore the molecular mechanisms of pemetrexed involvedin cell growth, we used a two-dimensional polyacrylamide gel electrophoresis (2-DE) proteomics approach toanalyze proteins changed in A549 cells treated with pemetrexed. As a result, twenty differentially expressedproteins were identified by ESI-Q-TOF MS/MS analysis in A549 cells incubated with pemetrexed comparedwith non-treated A549 cells. Three key proteins (GAPDH, HSPB1 and EIF4E) changed in pemetrexed treatedA549 cells were validated by Western blotting. Accumulation of GAPDH and decrease of HSPB1 and EIF4Ewhich induce apoptosis through inhibiting phosphorylation of Akt were noted. Expression of p-Akt in A549 cellstreated with pemetrexed was reduced. Thus, pemetrexed induced apoptosis in A549 cells through inhibiting theAkt pathway.