To explore the mechanism and synergistic effect of vitamin A and vitamin D in inducing apoptosis of prostate cancer cells. The cell proliferation activity was determined by MTT assay. The proportion of apoptotic cells was analyzed by FACS and fluorescence intensity. TUNEL was used to evaluate vitamin A and vitamin D's induction of apoptosis in prostate cancer cells. The protein and mRNA expression level of Cyclin D1 and Bax were determined by real time-PCR and western blot. The results of MTT showed vitamin A and vitamin D's inhibition on proliferation ratio in prostate cancer cells is time and concentration dependent. FACS and fluorescence intensity analysis proved that the proportion of apoptotic cells increased after vitamin A and vitamin D treatment. TUNEL showed vitamin A and vitamin D induced prostate cancer cells apoptosis. The combination of vitamin A and vitamin D markedly enhanced the expression of Bax and reduced the expression of Cyclin D1 by real time-PCR and western blot assay. In conclusion, vitamin A and vitamin D could synergistically induce apoptosis in prostate cancer cells.