The immunosuppressive microenvironment in pancreatic cancer, characterized by low tumor-specific T cells and excessive fibrosis, limits the effectiveness of immunotherapy. Here, three datasets and multi-immunofluorescence staining of tissue microarrays in pancreatic cancer indicate that mesothelin (MSLN) expression negatively correlates with cytotoxic T cells in tumor. Anti-MSLN antibody (αMSLN) treatment of pancreatic cancer in vivo can significantly increase T cell infiltration. Meanwhile, the combination of αMSLN and neoantigen peptide vaccine identified from pancreatic cancer cell lines is demonstrated to be more effective in inducing neoantigen-specific T cell generation and infiltration at subcutaneous and orthotopic pancreatic cancer models for enhancing antitumor efficacy. Single-cell transcriptome analysis shows that the combined treatment significantly reduces the proportion of fibroblasts, improves the infiltration of IFN-γCD4 and GZMKCD8 T cells, as well as reduces the interaction of antigen presentation-associated ligands and receptors between antigen-presenting Cancer-Associated Fibroblasts (apCAFs) and naive CD4 T cells. The negative correlations between apCAFs and CD8 T cells/IFN-γCD4 T cells are further confirmed in human pancreatic cancer tissues. Overall, this study demonstrates that targeting MSLN can improve neoantigen vaccine induced immune efficacy by reducing apCAFs to interrupt the conversion of naive CD4 T cells to Tregs, and therefore increase the infiltration of tumor-specific T cells.