Type 2 melastatin-related transient receptor potential channel (TRPM2), a member of the melastatin-related TRP (transient receptor potential) subfamily is a Ca2+-permeable channel activated by hydrogen peroxide (H2O2). We have investigated the role of TRPM2 channels in mediating the H2O2-induced increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i) in insulin-secreting cells. In fura-2 loaded INS-1E cells, a widely used model of β-cells, and in human β-cells, H2O2 increased [Ca2+]i, in the presence of 3 mM glucose, by inducing Ca2+ influx across the plasma membrane. H2O2-induced Ca2+ influx was not blocked by nimodipine, a blocker of the L-type voltage-gated Ca2+ channels nor by 2-aminoethoxydiphenyl borate, a blocker of several TRP channels and store-operated channels, but it was completely blocked by N-(p-amylcinnamoyl)anthranilic acid (ACA), a potent inhibitor of TRPM2. Adenosine diphosphate phosphate ribose, a specific activator of TRPM2 channel and H2O2, induced inward cation currents that were blocked by ACA. Western blot using antibodies directed to the epitopes on the N-terminal and on the C-terminal parts of TRPM2 identified the full length TRPM2 (TRPM2-L), and the C-terminally truncated TRPM2 (TRPM2-S) in human islets. We conclude that functional TRPM2 channels mediate H2O2-induced Ca2+ entry in β-cells, a process potently inhibited by ACA.