The mechanisms by which mitochondria regulate the sustained phase of agonist-induced responses in cytosolic Ca2+ concentration as an independent organelle in whole is not clear. By exposing to ethidium bromide and supplying pyruvate and uridine, we established mitochondrial DNA (mtDNA)-depleted rat airway smooth muscle cells (RASMCs) with maintained cellular energy. Upon an exposure to 2 μM histamine, [Ca2+]i in control RASMCs increased to a peak followed by a plateau above baseline, whereas [Ca2+]i in mtDNA-depleted RASMCs jumped to a peak and then declined to baseline without any plateau. mtDNA depletion apparently attenuated intracellular reactive oxygen species generation induced by histamine. By coexposure to 2 μM histamine and 0.1 μM exogenous H2O2, which did not affect [Ca2+]i by itself, the above difference in [Ca2+]i kinetics in mtDNA-depleted RASMCs was reversed. Intracellular H2O2 decomposition abolishes histamine-induced sustained elevation in [Ca2+]i in RASMCs. Thus, mitochondria regulate agonist-induced sustained [Ca2+]i elevation by a H2O2-dependent mechanism.