Prostate stem-like cells exhibit resistance to anti-androgen agents and contribute to the recurrence of this cancer. A novel approach to overcome the resistance of prostate cancer stem-like cells is thus essential. The present study investigated whether soybean derived Bowman-Birk inhibitor (BBI) could abrogate the resistance of prostate cancer stem-like cells from an androgen-dependent prostate cancer cell line (LNCaP cells) to an anti-androgen agent. The LNCaP stem-like cells exhibited stronger resistance to an androgen antagonist, flutamide, than LNCaP parental cells. Exposure of LNCaP stem-like cells to BBI treatment reduced their stemness and resistance to flutamide. In parallel, LNCaP stem-like cells showed a positive response based on changes in the typical androgen-governed gene, prostate-specific antigen. These results suggest the potential of BBI as a candidate to reduce the resistance of prostate cancer stem-like cells to antiandrogen agents.