Oxidative stress through the production of oxygen metabolites such as hydrogen peroxide (H₂O₂) increases vascular endothelial permeability. H₂O₂ stimulates ADP-ribose formation, which in turn opens transient receptor potential melastatin (TRPM)2 channels. Here, in endothelial cells, we demonstrate transcript and protein expression of TRPM2, a Ca²⁺-permeable, nonselective cation channel. We further show the importance of TRPM2 expression in signaling of increased endothelial permeability by oxidative stress. Exposure of endothelial cell monolayers to sublytic concentrations of H₂O₂ induced a cationic current measured by patch-clamp recording and Ca²⁺ entry detected by intracellular fura-2 fluorescence. H₂O₂ in a concentration-dependent manner also decreased trans-monolayer transendothelial electrical resistance for 3 hours (with maximal effect seen at 300 μmol/L H₂O₂), indicating opening of interendothelial junctions. The cationic current, Ca²⁺ entry, and transendothelial electrical resistance decrease elicited by H₂O₂ were inhibited by siRNA depleting TRPM2 or antibody blocking of TRPM2. H₂O₂ responses were attenuated by overexpression of the dominant-negative splice variant of TRPM2 or inhibition of ADP-ribose formation. Overexpression of the full-length TRPM2 enhanced H₂O₂-mediated Ca²⁺ entry, cationic current, and the transendothelial electrical resistance decrease. Thus, TRPM2 mediates H₂O₂-induced increase in endothelial permeability through the activation of Ca²⁺ entry via TRPM2. TRPM2 represents a novel therapeutic target directed against oxidant-induced endothelial barrier disruption.