© 2016, Springer Science+Business Media New York.The Zn2+ in cardiomyocytes is buffered by structures near T-tubulus and/or sarcoplasmic/endoplasmic reticulum (S(E)R) while playing roles as either an antioxidant or a toxic agent, depending on the concentration. Therefore, we aimed first to examine a direct effect of ZnPO4 (extracellular exposure) or Zn2+ pyrithione (ZnPT) (intracellular exposure) application on the structure of the mitochondrion in ventricular cardiomyocytes by using histological investigations. The light microscopy data demonstrated that Zn2+ exposure induced marked increases on cellular surface area, an indication of hypertrophy, in a concentration-dependent manner. Furthermore, a whole-cell patch-clamp measurement of cell capacitance also supported the hypertrophy in the cells. We observed marked increases in mitochondrial matrix/cristae area and matrix volume together with increased lysosome numbers in ZnPO4- or ZnPT-incubated cells by using transmission electron microscopy, again in a concentration-dependent manner. Furthermore, we observed notable clustering and vacuolated mitochondrion, markedly disrupted and damaged myofibrils, and electron-dense small granules in Zn2+-exposed cells together with some implications of fission-fusion defects in the mitochondria. Moreover, we observed marked depolarization in mitochondrial membrane potential during 1-μM ZnPT minute applications by using confocal microscopy. We also showed that 1-μM ZnPT incubation induced significant increases in the phosphorylation levels of GSK3β (Ser21 and Ser9), Akt (Ser473), and NFκB (Ser276 and Thr254) together with increased expression levels in ER stress proteins such as GRP78 and calregulin. Furthermore, a new key player at ER-mitochondria sites, promyelocytic leukemia protein (PML) level, was markedly increased in ZnPT-incubated cells. As a summary, our present data suggest that increased cytosolic free Zn2+ can induce marked alterations in mitochondrion morphology as well as depolarization in mitochondrion membrane potential and changes in some cytosolic signaling proteins as well as a defect in ER-mitochondria cross talk.