Apoptosis contributes to the regulation of cell growth and regeneration and to the development of neoplasia. Mcl-1 is an anti-apoptotic protein that is particularly important for the development of hematological and biliary malignancies, but the mechanism of action of Mcl-1 is unknown. A number of pro- and anti-apoptotic proteins exhibit their effects by modulating Ca2+ signals, so we examined the effects of Mcl-1 on components of the Ca2+ signaling pathway that are known to regulate apoptosis. Expression of Mcl-1 did not affect expression of the inositol 1,4,5-trisphosphate receptor or the size of endoplasmic reticulum Ca2+ stores. However, mitochondrial Ca2+ signals induced by either Ca2+ agonists or apoptotic stimuli were decreased in cells overexpressing Mcl-1 and increased in cells in which Mcl-1 expression was inhibited. These findings provide evidence that Mcl-1 directly inhibits Ca2+ signals within mitochondria, which may provide a novel mechanism to inhibit apoptosis and thereby promote neoplasia.