Besides their prominent function in cellular energy metabolism, the central role of mitochondria has been focused on control of cellular death in last decades. The mitochondrial permeability transition pore (PTP) is involved in the intrinsic pathway of apoptosis via the release of cytochrome c into cytosol. Metformin, a drug widely used in the treatment of type II diabetes, has recently received attention owing to new findings regarding its effect on apoptosis through mitochondrial permeability transition and cytochrome c release. The modulation of PTP is still unknown, but calcium is certainly the most important known inducer. In the present study, the preventive effects of metformin on gentamicin ototoxicity were investigated through the changes of intracellular calcium concentrations using calcium imaging in HEI-OC1 cells. Calcium imaging traced the changes of intracellular calcium concentration after the application of 50 mM of gentamicin in both 100 uM of metformin pretreated group and non-pretreated group. These calcium reactions were compared and analyzed with the results of cell viability test, Hoechst staining, intracellular reactive oxygen species level and expression of caspase-3, and poly-ADP-ribose polymerase (PARP). Continuous increase of intracellular calcium concentration (increase of 380/340 ratio) occurred after application of 50 mM of gentamicin. However, there was no change of intracellular calcium concentration in 100 uM metformin pretreated group. Cell viability was significantly higher in 100 uM metformin pretreated group and also, metformin pretreated HEI-OC1 cells produced less ROS that gentamicin alone treated group. Gentamicin increased cleaved PARP and caspase-3, but metformin inhibited the expression of caspase-3 and cleavage of PARP. This study demonstrated that metformin prevented gentamicin induced apoptosis through the calcium modulating and ROS reducing anti-apoptotic effects.