The treatment of castration-resistant prostate cancer (CRPC) is always a difficulty in the clinic. Most patients with localized tumor eventually develop CRPC, even if hormone therapy is initially effective. Increasing evidence shows immunotherapy has special advantages compared with traditional therapy in cancer treatment. In this study, we constructed the DC-PC-3 fusion vaccine with B7-1- and GM-CSF-specific modification, and studied its ability to stimulate specific immune response and anti-tumor effect in vitro. The results showed that fusion of DC and tumor cells can improve the expression of associated antigens of DCs. DC-tumor fusion vaccine can strongly promote T cell proliferation and IFN-γ secretion and induce a significant tumor-specific cytotoxic T lymphocyte response. In addition, the B7-1/GM-CSF-modified fusion vaccine showed a more significant anti-tumor effect and greater ability to stimulate the immune response than that without specific modification in vitro. Thus, GM-CSF/B7-1-modified fusion vaccine might be used as a potential therapy strategy for prostate cancer.