Objective: To explore the effects of over-expression of GPC5 on the biological function of human lung adenocarcinoma A549 cancer-initiating cells(CICs). Methods: Lentivims-induced GPC5 over-expression was used for biological functional analyses in human lung adenocarcinoma A549 cell line, including cancer-initiating cell microspherescounting by light microscopy, CD133 positive cells measured by flow cytometry, migration and invasive activity detected by Matrigel and Transwell assay, as well as epithehal-mesenchymal transition-related protein expression detected by Western blot. Results: Compared with the control group, the numbers of cancer-initiating cell microspheres remarkly decreased(P < 0.05) and the percent of CDl33 positive cells was reduced (P < 0.05) in the GPC5 over-expressed group. The over-expression of GPC5 also inhibited the invasion and migration ability of CICs, and led to the down-regulation of Vimentin and N-cadherin, as well as the up-regulation of E-cadherin. Conclusion: The over-expression of GPC5 can inhibit the genesis, invasion and migration of human lung adenocarcinoma A549-CICs, and regulate the expression of EMT related proteins.