To discover novel anticancer agents with potent and low toxicity, we designed and synthesized a range of new thiosemicarbazone-indole analogues based on lead compound 4 we reported previously. Most compounds displayed moderate to high anticancer activities against five tested tumor cells (PC3, EC109, DU-145, MGC803, MCF-7). Specifically, the represented compound 16f possessed strong antiproliferative potency and high selectivity toward PC3 cells with the IC₅₀ value of 0.054 μM, compared with normal WPMY-1 cells with the IC₅₀ value of 19.470 μM. Preliminary mechanism research indicated that compound 16f could significantly suppress prostate cancer cells (PC3, DU-145) growth and colony formation in a dose-dependent manner. Besides, derivative 16f induced G1/S cycle arrest and apoptosis, which may be related to ROS accumulation due to the activation of MAPK signaling pathway. Furthermore, molecule 16f could effectively inhibit tumor growth through a xenograft model bearing PC3 cells and had no evident toxicity in vivo. Overall, based on the biological activity evaluation, analogue 16f can be viewed as a potential lead compound for further development of novel anti-prostate cancer drug.