Invadopodia are detected in various types of human cancer and Src-transformed fibroblasts. It is unclear, however, whether the invadopodia involved in lung cancer invasion and metastasis. To find out the presence of invadopodia in lung cancer cells and the regulation mechanisms of invadopodia, we carried out immunofluorescence and FITC-gelatin degradation assay and transplanted lung cancer mouse model to demonstrate the presence of invadopodia in human lung cancer cell lines, L9981, NL9980 and A549. In addition, we detected the expression of osteopontin, cortactin, Arp2, and MMP9 in NL9980 and NL9980-OPN. Our investigations indeed demonstrated that invadopodia are present in lung cancer cells A549 and L9981. Invadopodia enable increased invasion and metastasis of A549 and L9981 cells . Moreover, A549 and NL9980 cells forced overexpressing osteopontin (OPN) displayed an increase in the number of invadopodia and gelatinolytic activity accompany with elevated expression of cortactin, Arp2, in addition, OPN can remarkably up-regulated the expression of cortactin, Arp2 and MMP9 of A549 cells in a time-dependent manner. Furthermore, we found that forced overexpressing of OPN activate the Src, ERK1/2 phosphorylation. Thus, we conclude that invadopodia participates in the process of invasion and metastasis of human lung cancer, and OPN can improve the invadopodia formation by activate the Src, ERK1/2 pathway.