The development of metastasis is the leading cause of death and an enormous therapeutic challenge in cases of non-small cell lung cancer. To better understand the molecular mechanisms underlying the metastasis process and to discover novel potential clinical markers for non-small cell lung cancer, comparative proteomic analysis of two non-small cell lung cancer cell lines with different metastatic potentials, the non-metastatic CL1-0 and highly metastatic CL1-5 cell lines, was carried out using two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and tandem mass spectrometry. Thirty-three differentially expressed proteins were identified unambiguously, among which 16 proteins were significantly upregulated and 17 proteins were downregulated in highly metastatic CL1-5 cells compared with non-metastatic CL1-0 cells. Subsequently, 8 of 33 identified proteins were selected for further validation at the mRNA level using real-time quantitative polymerase chain reaction, and three identified proteins, S100A11, PGP 9.5 and HSP27, were confirmed by western blotting. The protein S100A11 displaying significant differential expression at both the protein and mRNA levels was further analyzed by immunohistochemical staining in 65 primary non-small cell lung cancer tissues and 10 matched local positive lymph node specimens to explore its relationship with metastasis. The results indicated that the upregulation of S100A11 expression in non-small cell lung cancer tissues was significantly associated with higher tumor-node-metastasis stage (P = 0.001) and positive lymph node status (P = 0.011), implying that S100A11 might be an important regulatory molecule in promoting invasion and metastasis of non-small cell lung cancer.