Reactive oxygen species, including hydrogen peroxide (H₂O₂), are known to induce β-cell apoptosis. The present study investigated the role of Ca²⁺ in H₂O₂-induced apoptosis of the β-cell line INS-1. Annexin V assay with flow cytometry and DNA ladder assay demonstrated that treatment of INS-1 cells with 100 μM H₂O₂ for 18 h significantly increased apoptotic cells. A comparable level of apoptosis was also observed after 18 h when the cells were treated with 100 μM H₂O₂ only for initial 30 min. The H₂O₂-induced apoptosis was abolished by 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA/AM), a chelator of intracellular Ca²⁺, by 2-aminoethoxydiphenylborate (2-APB), a blocker of inositol 1,4,5-trisphosphate (IP₃) receptors and cation channels, and by xestospongin D, a blocker of IP₃ receptors, and was partially blocked by SKF-96365, a non-selective cation channel blocker. However, nicardipine, an L-type voltage-dependent Ca²⁺ channel blocker, or N-(p-amylcinnamoyl)anthranilic acid (ACA), a TRPM2 blocker, had little effect on the apoptosis. The inhibitory effect of BAPTA/AM or 2-APB on the H₂O₂-induced apoptosis was largely attenuated when the drug was added 30 min or 1 h after start of the treatment with H₂O₂. These results suggest that the initial intracellular Ca²⁺ elevation induced by H₂O₂, which is mediated via IP₃ receptors and store-operated cation channels, plays an obligatory role in the induction of β-cell apoptosis.