Objective:To construct the second and fourth generations of CAR-T cells targeting fibroblast activation protein (FAP) on the surface of stromal carcinoma-associated fibroblasts and compare their characteristics in vitro and in vivo. Methods:ELISA was used to detect the cytokines secreted by CAR-T cells. Cell proliferation and viability were analyzed by counting. Chemotactic ability was tested by Transwell migration assay. Distribution of T cell subsets was analyzed by flow cytometry. Cytotoxicity was assessed by luciferase bioluminescence. The safety and therapeutic effects were evaluated in a NOG mouse model of metastatic human lung cancer.Results:The expression rates of the second and fourth generations of CAR-T cells (h4BBz CAR-T and h4BBz-7.19 CAR-T) were (74.280±4.384)% and (67.220±4.013)%, respectively. The h4BBz-7.19 CAR-T cells had better in vitro proliferation and chemotactic activity than h4BBz CAR-T cells as they were able to secrete IL-7 and CCL19, while the viability of h4BBz-7.19 CAR-T cells was comparable to that of h4BBz CAR-T cells. There was no significant difference in the expression rate of h4BBz CAR or h4BBz-7.19 CAR between CD4 + T and CD8 + T cells. The proportions of both Naive cells and T memory stem cells (TSCM) in CD4 + and CD8 + T cells were higher in h4BBz-7.19 CAR-T cells than in h4BBz CAR-T cells. Moreover, h4BBz-7.19 CAR-T cells possessed stronger specific cytotoxicity on the target cancer cells than h4BBz CAR-T cells when the ratio of effectors/targets was low ( P1∶1=0.004, P2∶1=0.000 6, P5∶1<0.000 1, P10∶1=0.022, P20∶1=0.116), while the expression of PD-1 on the surface of h4BBz-7.19 CAR-T cells was lower than that on h4BBz CAR-T cells. In the NOG mouse model of metastatic human lung cancer, h4BBz-7.19 CAR-T cells could slow the tumor growth and prolong the survival time of mice without causing weight loss or pathological changes in the organs. Conclusions:The fourth-generation CAR-T cells targeting FAP were shown to have stronger proliferation, better penetration and more potent specific cytotoxicity by secreting IL-7 and CCL19 and could slow the tumor growth and prolong survival by improving tumor immunosuppressive microenvironment. This study provided reference for the clinical application of the fourth generation of CAR-T cells.