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Prostate cancer is a leading cause of cancer-associated death in males. Leonurine (Leo) is a pleiotropic anti-tumor agent isolated from traditional Chinese herb that was used in gynecologic treatments. However, its pharmacological effect against prostate cancer progression remains unclear. Here, we showed that Leo dose dependently inhibited prostate cancer cell proliferation, promoted cell apoptosis, and induced cell cycle arrest. Moreover, we noticed that miR-18a-5p was downregulated and the solute carrier family 40 member 1 (SLC40A1) is upregulated by Leo treatment. SLC40A1 knockdown by siRNA abrogated the inhibitory effect of Leo on prostate cancer progression. Notably, Leo also significantly inhibited prostate cancer progression in a subcutaneous xenograft tumor mouse model in vivo. This study further unveiled the mechanism by which Leo inhibited prostate cancer progression, which provides a promising potential for its future clinical application.