To discover metastasis-associated proteins within cancer cells, we used the isobaric tags for relative and absolute quantitation (iTRAQ) approach combined with nano liquid chromatography-tandem mass spectrometry (NanoLC-MS/MS) analysis to identify proteins that were differentially expressed between lung adenocarcinoma cancer cell lines SPC-A-1sci cells with high metastatic potential and parent SPC-A-1 cells with low metastatic potential. By employing biological and technical replicates, we identified 5818 nonredundant proteins and quantified 5443 proteins, 256 of which were differentially expressed in the two cell lines. Through si-RNA-mediated functional screens, Myosin heavy chain 9 (MYH9) and Copine III (CPNE3) were indicated as positively correlating with the migration and invasion properties of SPC-A1sci cells, and the same function of CPNE3 was confirmed in another lung cancer cell line, H1299. Furthermore, overexpressing CPNE3 promoted nonsmall-cell lung cancer (NSCLC) cell line (SPC-A-1 and XL-2) migration and invasion in vitro. Moreover, the targeted knock-down of CPNE3 inhibited the in vivo metastatic abilities of H1299 cells in mouse models. Lastly, immunohistochemistry revealed that the CPNE3 expression level was positively correlated with the clinical stage and TNM classification in NSCLC patients. Taken together, our results indicate that CPNE3 could play a critical role in NSCLC metastasis.