SUMO-specific protease 1 (SENP1), a member of the de-SUMOylation protease family, is elevated in prostate cancer (PCa) cells and is involved in PCa pathogenesis. Momordin Ιc (Mc), a natural pentacyclic triterpenoid, inhibited SENP1 in vitro, as reflected by reduced SENP1C-induced cleavage of SUMO2-ΔRanGAP1. Mc also altered the thermal stability of SENP1 in a newly developed cellular thermal shift assay, indicating that Mc directly interacts with SENP1 in PCa cells. Consistent with SENP1 inhibition, Mc increased SUMOylated protein levels, which was further confirmed by the accumulation of two known SUMOylated proteins, hypoxia inducible factor-1a and nucleus accumbens associated protein 1 in PC3 cells. Compared to LNCaP and normal prostate epithelial RWPE-1 cells, PC3 cells had higher levels of SENP1 mRNA and were more sensitive to Mc-induced growth inhibition. Mc also reduced SENP1 mRNA levels in PCa cells. Overexpression of SENP1 rescued PC3 cells from Mc-induced apoptosis. Finally, Mc suppressed cell proliferation and induced cell death in vivo in a xenograft PC3 tumor mouse model. These findings demonstrate that Mc is a novel SENP1 inhibitor with potential therapeutic value for PCa. Investigation of other pentacyclic triterpenoids may aid in the development of novel SENP1 inhibitor drugs.