Upregulation of mitochondrial function and oxidative metabolism is a hallmark in the differentiation of stem cells. However, the mechanism underlying the metabolic reprogramming and upregulation of mitochondrial function during the differentiation of human mesenchymal stem cells (hMSCs) is largely unclear. Sirt3 has emerged as a sensor in regulating mitochondrial function and antioxidant defence system in cellular response to energy demand or environmental stimuli, but its roles in stem cell differentiation have not been fully understood. In this study, we used adipose-derived hMSCs (ad-hMSCs) to investigate the role of Sirt3 in adipogenic differentiation and in the function of mature adipocytes. We showed that at the early stage of adipogenic differentiation, Sirt3 upregulation is essential for the activation of biogenesis and bioenergetic function of mitochondria. In addition, we found that induction of Forkhead Box O 3a (FoxO3a), an upstream factor that regulates MnSOD gene transcription, is involved in the upregulation of antioxidant enzymes at the early stage of adipogenic differentiation. Silencing of Sirt3 by shRNA decreased the protein level of FoxO3a and subsequently downregulated a number of FoxO3a-mediated antioxidant enzymes and increased oxidative stress in ad-hMSCs after adipogenic induction. Importantly, depletion of Sirt3 compromised the ability of ad-hMSCs to undergo adipogenic differentiation and led to adipocyte dysfunction and insulin resistance. These findings suggest that Sirt3-mediated protein deacetylation plays an important role in regulating oxidative metabolism and antioxidant defence in stem cell differentiation, and that Sirt3 deficiency may be related to insulin resistance.Keywords: Adipogenesis; Sirt3; antioxidant defence; human mesenchymal stem cells; insulin resistance; mitochondrial bioenergetics.