A novel therapeutic vaccine against prostate cancer was developed by simultaneous immobilization of streptavidin-tagged bioactive GM-CSF and TNFα on the biotinylated surface of 30% ethanol-fixed RM-1 prostatic cancer cells. This study showed that the GM-CSF/TNFα-doubly surface-modified vaccine significantly extended the survival in the orthotopic model of RM-1 prostate cancer, and was superior to single GM-CSF- or TNFα-surface-modified vaccine. Moreover, the splenocytes from the GM-CSF/TNFα-vaccine-treated mice showed the most potent cytotoxicity on RM-1 cells and the highest production of RM-1-specific IFNγ. In addition, more CD4 + and CD8 + T cells infiltrated into the tumor sites in the GM-CSF/TNFα-vaccine-treated mice than in the GM-CSF- or TNFα-vaccine-treated mice. Therefore, our study demonstrated that the efficacy of RM-1 prostate cancer cell vaccine could be improved by conjugating both GM-CSF and TNFα simultaneously on the surface of cancer cells, and that this modification thus has a potential translational significance.