Hormone antagonists can be effective tools to delineate receptor signaling pathways and their resulting downstream physiological actions. Mutation of the receptor binding domain (RBD) of human H2 relaxin (H2) impaired its biological function as measured by cAMP signaling. In a competition assay, H2 exhibited antagonistic activity by blocking recombinant H2 relaxin from binding to receptors on THP-1 cells. In a flow cytometry-based binding assay, H2 demonstrated weak binding to 293T cells expressing the LGR7 receptor in the presence of biotinylated H2 relaxin. When human prostate cancer cell lines (PC-3 and LNCaP) were engineered to overexpress eGFP, wild-type (WT) H2, or H2, and subsequently implanted into NOD/SCID mice, tumor xenografts overexpressing H2 displayed smaller volumes compared to H2 and eGFP controls. Plasma osmolality readings and microvessel density and area assessment suggest that H2 modulates physiological parameters in vivo. In a second murine model, intratumoral injections of lentivectors engineered to express H2/eGFP led to suppressed tumor growth compared to controls. This study provides further evidence supporting a role for H2 relaxin in prostate tumor growth. More importantly, we report how mutation of the H2 relaxin RBD confers the hormone derivative with antagonistic properties, offering a novel reagent for relaxin research.--Silvertown, J. D., Symes, J. C., Neschadim, A., Nonaka, T., Kao, J. C. H., Summerlee, A. J. S., Medin, J. A. Analog of H2 relaxin exhibits antagonistic properties and impairs prostate tumor growth.