Transient receptor potential melastatin 2 (TRPM2) is a calcium-permeable channel activated by adenosine diphosphate ribose metabolites and oxidative stress. TRPM2 contributes to neuronal injury in the brain caused by stroke and cardiac arrest among other diseases including pain, inflammation, and cancer. However, the lack of specific inhibitors hinders the study of TRPM2 in brain pathophysiology. Here, we present the design of a novel TRPM2 antagonist, tatM2NX, which prevents ligand binding and TRPM2 activation. We used mutagenesis of tatM2NX to determine the structure-activity relationship and antagonistic mechanism on TRPM2 using whole-cell patch clamp and Calcium imaging in human embryonic kidney 293 cells with stable human TRPM2 expression. We show that tatM2NX inhibits over 90% of TRPM2 channel currents at concentrations as low as 2 μM. Moreover, tatM2NX is a potent antagonist with an IC of 396 nM. Our results from tatM2NX mutagenesis indicate that specific residues within the tatM2NX C terminus are required to confer antagonism on TRPM2. Therefore, the peptide tatM2NX represents a new tool for the study of TRPM2 function in cell biology and enhances our understanding of TRPM2 in disease. SIGNIFICANCE STATEMENT: TatM2NX is a potent TRPM2 channel antagonist with the potential for clinical benefit in neurological diseases. This study characterizes interactions of tatM2NX with TRPM2 and the mechanism of action using structure-activity analysis.