||The calcium ion (Ca2+), as an important second messenger, is known to be involved in many cellular functions. The present study is aimed at investigating the roles of Ca2+ signaling in regulating apoptosis of mammalian cells. We tried to answer the following questions: (1) Is the mitochondrial Ca2+ signal involved in the early stage to drive the progression of apoptosis? (2) What are the characteristics and transportation mechanisms of the mitochondrial Ca2+ signal in apoptosis? (3) What are the mechanisms by which the Bcl-2 family proteins such as Bcl-2 and Bax regulate the Ca2+ mobilization from endoplasmic reticulum (ER) to cytosol and mitochondria during apoptosis? (4) Are there any other regulators such as Bcl-2 family proteins binding partners affecting the Ca2+ signals? Using single living cell Ca2+ imaging techniques, we found that mitochondrial Ca2+ signal plays a positive role in the early stage of UV- or TNFα- induced apoptosis in HeLa cells. The transient mitochondrial Ca2+ spikes are synchronous with the cytosolic Ca2+ changes. Our results suggested that the Ca2+ release from ER plays an important role on regulating the apoptotic process since the cytosolic Ca2+ elevation during ATP treatment and TNFα-induced apoptosis are highly correlated. We then investigated the mechanisms by which Bcl-2 family proteins affect the IP3 dependent Ca2+ mobilization. We found that pro-apoptotic protein Bax can counteract with anti-apoptotic Bcl-2 to facilitate the ER Ca2+ release by interacting with IP3 receptor. Our results suggested that Bax could sensitize the IP3 receptor, while Bcl-2 could inhibit IP3 receptor during apoptosis. Finally, we examined the effect of other Bcl-2 family proteins and their binding partners on Ca2+ signaling. Our results indicated that the Bcl-2 family proteins may regulate the apoptotic process through modulating the Ca2+ mobilization from ER to cytosol and mitochondria.