BACKGROUND: Prostate cancer stem cell is an important reason for the invasion and recurrence of prostatic carcinoma. However, separation efficiency of prostate cancer stem cells is very low.OBJECTIVE: To explore the efficient method for isolating and identifying the prostate cancer stem cells from human prostatic carcinoma cell lines PC-3 and LNCap. METHODS: Prostate cancer cell lines PC-3 and LNCap were cultured in serum free medium (SFM) and serum supplemented medium (SSM), respectively. The percentage of prostate cancer stem cells from different medium was detected by flow cytometry through markers CD133 and CD44, and the properties of prostate cancer stem cells were preliminarily identified using inducing differentiation experiments.RESULTS AND CONCLUSION: PC-3 and LNCap formed sphere cells in SFM, which can be induced into adherent cells after culture in SSM. Higher percentage of CD44+/CD133+cells was obtained from LNCap cells (1.71%; 0.73%) than PC-3 cells (0.59%; 0.32%) in both SFM and SSM. The number of CD44+/CD133+ LNCap cells was more than PC-3 using both methods (P < 0.05), but the efficiency of SFM and SSM had no statistical significance (P > 0.05). However, the culture cycle was longer and number of obtained cells was less by SFM culture, directly influencing functional determination of prostate cancer stem cells. Compared with suspension culture method with SFM, SSM is more convenient and effective in isolating prostate cancer stem cells from LNCap cells.