Introduction Mitochondria are supplying the cell with adenosine triphosphate (ATP) and are crucially involved in cell differentiation, cell cycle and intracellular signaling. Since they are essential for cell survival, their important role in human diseases and aging processes is under intensive investigation. The transfer of Ca2+ into the matrix of mitochondria is an important process that substantially contributes to multiple cellular functions. The Ca2+ concentration within mitochondria ([Ca2+]mito) controls, for instance, the metabolic activity of these organelles and can also trigger cell death pathways. Methods To investigate the role and possible mechanism(s) of cell death pathways triggered by mitochondrial Ca2+, we applied resveratrol and its derivative, piceatannol, which are known to cause apoptosis in certain cancer cells. Using genetically encoded, mitochondria or endoplasmic reticulum (ER) targeted, fluorescent Ca2+ and ATP probes as well as fluorescent dyes, we investigated how these compounds affect calcium and ATP homeostasis in HeLa and Ea.hy926 in comparison to freshly isolated human umbilical vein endothelial cells (HUVECs). Hence, we studied the effect of these compounds on cell viability by MTT assay in tumor and somatic cells. Results Our results indicated that chemical compounds such as resveratrol, piceatannol and oligomycin bind different subunits of the ATP synthase and reduce mitochondrial ATP production. The lack of sufficient ATP supply in the gap between mitochondria and the ER reduced SERCA activity. This resulted in slower refilling of ER Ca2+ stores, an increased intra-organelle Ca2+ concentration and, consequently, an increased mitochondrial Ca2+ uptake upon IP3-mediated intracellular Ca2+ release. Such excessive mitochondrial Ca2+ uptake resulted in mitochondrial Ca2+ overload and ultimately cell death in cancer cells, while this effect was significantly reduced in somatic cells due to lower stability of mitochondria-associated ER membranes (MAMs). Conclusion Resveratrol and its derivative piceatannol act as new MitoCans by triggering cell death pathways via mitochondrial Ca2+ overload in cancer cells because of cancer-specific mitochondrial Ca2+ homeostasis with higher MAM stability than somatic cells. Moreover, this study reveals a crucial involvement of mitochondria in cancer cell survival. These organelles are therefore promising targets for future treatments