Osteoporosis is a bone disease that has been connected with reactive oxygen species (ROS)-induced cytotoxicity. Mitochondrial dysfunction may be involved in the mechanism underlying ROS-induced cytotoxicity. It has been demonstrated that melatonin may exert cytoprotective effects by improving mitochondrial energetics and functions in several models of oxidative damage. In the present study, the MG63 osteoblast-like cell line was exposed to different concentrations of hydrogen peroxide (H2O2; 0, 100, 200, 400 or 800 µM) for 8 h, and 200 or 400 µM H2O2 for various periods of time (0.5, 4, 8 or 12 h). Results showed that H2O2 significantly reduced cell viability, increased the release of lactate dehydrogenase, increased the levels of ROS and malondialdehyde, reduced the concentration of adenosine-5'-triphosphate, disrupted the mitochondrial membrane potential (ΔΨm) and decreased the mitochondrial DNA copy number in MG63 cells. However, pretreatment with melatonin effectively decreased all of these H2O2-induced changes in cytotoxicity and mitochondrial dysfunction in MG63 cells. The protective effects of melatonin may be attributed to its ability to maintain mitochondrial function in H2O2-treated cells. This study suggests that melatonin is a potential pharmacological agent for preventing ROS-induced bone loss in diseases such as osteoporosis.