Objective By comparing the biological characteristics among Lewis lung cancer cells (LLC), LLC orthotopic transplantation derivative cells (R1-LLC) and R1-LLC orthotopic transplantation derivative cells (R2-LLC), we evaluate their invasion and metastatic abilities in orthotopic transplantation models. Methods In vitro, we applied CCK8, clonogenic assay, and Transwell invasion assay to detect the proliferation ability, invasion ability and morphologic structures of LLC,R1-LLC, R2-LLC cells respectively, meanwhile observing morphologic structures by transmission electron microscopy. In vivo, we constructed LLC, R1-LLC and R2-LLC orthotopic transplantation models. Herein, we observed their tumor growth and metastasis. The tumor formation rate and tumor-forming time were recorded for statistic analysis. Results In vitro: LLC, R1-LLC and R2-LLC cells showed no significant difference in proliferation ability(P> 0.05), but significant differences in invasion ability: R2-LLC> R1-LLC> LLC(P<0.05). In vivo: In the orthotopic group, the tumor formation rates of LLC, R1-LLC and R2-LLC cells were 66.67%、80%、93.33%(P <0.05). Conclusions In orthotopic implantation mouse models, R2-LLC cells present higher invasion and metastatic ability than R2-LLC and LLC cells.