To induce a potent cytotoxic T lymphocyte (CTL) response in dendritic cell (DC)-based immunotherapy against prostate cancer, various tumour antigens should be loaded onto DCs. The aim of this study was to establish a method of immunotherapy for castration-resistant prostate cancer (CRPC) using prostate cancer–specific CTLs generated in vitro by DCs. Monocyte-derived DCs from patients with CRPC were induced to mature using a standard cytokine cocktail (in IL-1β, TNF-α, IL-6 and PGE2: standard DCs, sDCs) or using an α-type 1-polarized DC (αDC1) cocktail (in IL-1β, TNF-α, IFN-α, IFN-γ and polyinosinic:polycytidylic acid) and loaded with the UVB-irradiated CRPC cell line PC-3. Antigen-loaded DCs were evaluated by morphological and functional assays. The αDC1s significantly increased the expression of several molecules related to DC maturation, regardless of whether the αDC1s were loaded with tumour antigens or not, compared to sDCs. The αDC1s showed a higher production of interleukin-12 both during maturation and after subsequent stimulation with CD40L, which was not significantly affected by loading with tumour antigens, as compared to standard DCs (sDCs). Prostate cancer–specific CTLs against autologous CRPC cells were successfully induced by αDC1s loaded with dying PC-3 cells. Autologous αDC1s loaded with an allogeneic CRPC cell line can generate greater CRPC-specific CTL responses as compared to sDCs and may provide a novel source of DC-based vaccines that can be used for the development of immunotherapy in patients with CRPC.