Osteosarcoma (OS) is the most common solid tumor of the bone that most often affects adolescents. The introduction of chemotherapy for the treatment of OS has largely improved the survival rates of patients with localized tumors. However, the 5-year survival rate of OS patients with relapsed or metastatic disease is only 10 to 20%. In this study, the antimicrobial peptide tilapia piscidin 3 (TP3), isolated from Nile tilapia (Oreochromis niloticus), was treated to OS MG63 cells. Our findings showed that TP3 concentration as low as 1 μM induced significant inhibition of cell viability and increased DNA fragmentation, as determined by the MTT and TUNEL assays, respectively. The protein expression levels of cleaved caspases 3/9 were increased. An in situ live-cell time-lapse video and cell tomographic microscopy images showed cellular blebbing, shrinkage, nuclear fragmentation, and chromatin condensation, with the formation of beaded apoptopodia. Moreover, there were significant increase in the production of TP3-induced mitochondrial and cellular reactive oxygen species (ROS), as well as down-regulated mitochondrial oxygen consumption and extracellular acidification rates. Additionally, TP3 enhanced mitochondrial fission, whereas fusion was attenuated. Furthermore, after administration of the mitochondria targeted antioxidant mitoTempo, TP3-induced ROS oxidant levels and alterations in cleaved caspases 3/9 expression were rescued. TP3 promoted mitochondria-modulated intrinsic apoptosis through the induction of ROS production, activation of caspases 3/9, and the down-regulation of mitochondrial oxygen consumption and extracellular acidification rates, suggesting that TP3 has potential as an innovative alternative for OS treatment.