Objective SIRT3 is a NAD⁺-dependent protein deacetylase localized on mitochondria and deacetylates mitochondrial proteins. Free fatty acids (FFAs) can lead to insulin resistance and pancreatic β cell dysfunction. In this study, we investigated whether overexpression of sirt3 protects free fatty acid induced-β cell dysfunction. Methods To investigate role of sirt3 in lipotoxicity-induced β cell dysfunction, expression of sirt3 was induced by using adenovirus in pancreatic β cell line, NIT-1. To generate lipotoxicity, palmitic acid was added to the media. ATP and cell viability were measured to determine the effect of sirt3 on mitochondria dysfunction. To examine effect of sirt3 on palmitate-induced p44/42 MAPK activation, Western blot analysis was performed. To elucidate the effect of sirt3 on fatty acid oxidation, fatty acid oxidation rate was measured using 14C-palmiate. Results When cell were treated with palmitate, cell viability was reduced and this cytotoxic effect was attenuated by overexpression of sirt3. While treatment of palmitate significantly reduced ATP level in NIT-1 cells, intracellular ATP level was not decreased in the presence of palmitate when sirt3 was overexpressed. Overexprssion of sirt3 suppressed palmitate-induced phosphorylation of p44/42 MAPK and p38. In addition, fatty acid oxidation was increased by sirt3 overexpression. Conclusion Overexpression of sirt3 protects free fatty acid induced-β cell dysfunction.