Background: Colorectal cancer (CRC) is the third prevalent malignant tumor all over the world, and many cases are metastatic at diagnosis. Immunotherapy emerges a promising treatment in CRC. However, anti-PD-1 monotherapy alone has not yielded a clinical benefit in majority of these patients. Therefore, combination therapy is necessary to overcome this limitation. Previously, we have developed a FAPα-expressing tumor vaccine and demonstrated its anti-tumor effects by reversing of immunosuppressive tumor microenvironment (TME). In the current study, we hypothesized that combination therapy could enhance the effect of anti-PD1 therapy. Methods: FAPα-expressing CT26 cells were produced as previously described. To establish xenograft tumor model, CT26 cells were injected into left flank of BALB/c mice. Anti-PD-1 monoclonal antibodies(mAb) were injected intraperitoneally twice weekly up to 5 injections. FAPα-vaccines were injected subcutaneously around tumors weekly up to 3 injections. Histological sections of tumours were analysed by H&E staining and immunofluorescence. Flow cytometric analysis was done using an FACSCalibur (BD) and analyzed using FlowJo software (TreeStar). Gene expression profile in TME was analyzed by bulk RNA sequencing. Results: In mice xenograft CT26 tumor model, either anti-PD-1 immunotherapy or FAPα tumor cell vaccine produced suppression of CT26 tumor growth compared with the control group. However, combining anti-PD-1 mAb and FAPα-expressing tumor cell vaccine elicited the most remarkable anti-tumor effect and induced the most pronounced tumor growth suppression among all groups. In combination therapy group of the vaccine and PD-1 mAb, there were significantly more infiltrated lymphocytes and less FAPα+ fibroblasts. Flow cytometry demonstrated that the combination therapy promoted M1 macrophage polarization with a reduction of M2-like tumor associated macrophages (TAMs) and an increase of M1-like TAMs. This polarization was in consistent with the reduced level of IL-10 that is predominant cytokine secreted by M2 macrophages. A reduction of myeloid-derived suppressor cells (MDSCs) in the TME were also observed. Using bulk RNAseq we observed a set of immune related genes upregulated following combination therapy, including innate immune response, antigen presentation, lymphatic cells and recruitment and T cells activation. Decreased secretion of immune inhibitory chemokines (eg, CXCL12, IL-10, TGFβ) was also found by ELISA in tumors from mice treated with combination therapy. Conclusions: Our preliminary results suggested that combination therapy of FAPα-expressing tumor vaccine and anti-PD-1 therapy is a promising way to improve the efficacy of immunotherapy through modulation of immunosuppressive TME. In the future, more research is needed to further elucidate mechanism and clinical value of this combination approach.