Here we explored the fusion of dendritic cells (DCs), potent antigen-presenting cells that initiate primary immune responses, with cancer-associated fibroblasts (CAFs), which are a stromal component needed for tumor progression, with the aim of stimulating T cells to inhibit tumor growth. Dendritic cells from the bone marrow of BALB/c mice were co-cultured with CAFs from H22 mouse hepatoma cells. CAFs were found to express fibroblast activation protein and α-smooth muscle actin by flow cytometry, Western blotting and immunofluorescence. Polyethylene glycol was added to the co-culture medium to encourage fusion, and the ability of the resulting fusion cells to produce TNF-α, IL-1β, IL-6, and IL-12p70 was confirmed using ELISA. These fusion cells efficiently stimulated T lymphocytes in vitro, causing them to generate IFN-α and IFN-γ. T cells activated by DC/CAF fusion cells led to strong CTL response against CAFs in vitro. The activated T cells also inhibited growth of H22 xenografts in vivo. These results indicate that DC/CAF fusion cells show potential for stimulating T cells as a novel anti-tumor vaccine.