Our previous study using apoptosis analysis suggested that Ca²⁺ release through inositol 1,4,5-trisphosphate (IP₃) receptors and the subsequent Ca²⁺ influx through store-operated channels (SOCs) constitute a triggering signal for H₂O₂-induced β-cell apoptosis. In the present study, we further examined the obligatory role of early Ca²⁺ responses in β-cell apoptosis induction. H₂O₂ induced elevation of the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) consisting of two phases: an initial transient [Ca²⁺]_c elevation within 30 min and a slowly developing one thereafter. The first phase was almost abolished by 2-aminoethoxydiphenylborate (2-APB), which blocks IP₃ receptors and cation channels including SOCs, while the second phase was only partially inhibited by 2-APB. The inhibition by 2-APB of the second phase was not observed when 2-APB was added 30 min after the treatment with H₂O₂. 2-APB also largely inhibited elevation of the mitochondrial Ca²⁺ concentration ([Ca²⁺]_m) induced by H₂O₂ when 2-APB was applied simultaneously with H₂O₂, but not when applied 30 min after H₂O₂ application. In addition, 2-APB inhibited the release of mitochondrial cytochrome c to the cytosol induced by H₂O₂ when 2-APB was applied simultaneously with H₂O₂ but not 30 min post-treatment. H₂O₂-induced [Ca²⁺]_m elevation and cell death were not inhibited by Ru360, an inhibitor of the mitochondrial calcium uniporter (MCU). These results suggest that the H₂O₂-induced initial [Ca²⁺]_c elevation, occurring within 30 min and mediated by Ca²⁺ release through IP₃ receptors and subsequent Ca²⁺ influx through SOCs, leads to [Ca²⁺]_m elevation, possibly through a mechanism independent of MCU, thereby inducing cytochrome c release and consequent apoptosis.