Obesity is one of the most common metabolic diseases worldwide, which is characterized by the excessive accumulation of triglycerides in adipocytes. Emerging evidence demonstrated that macroautophagy and chaperone-mediated autophagy (CMA) regulate lipid mobilization to maintain energy balance. Sirtuin 3 (SIRT3), a mitochondrial NAD⁺-dependent deacetylase, is essential in regulating macroautophagy and lipid metabolism. The role of SIRT3 in regulating macroautophagy and CMA in adipocytes is still unrevealed. In the current study, macroautophagy was found to be dynamically regulated during 3T3-L1 adipocytes differentiation, which coincided with SIRT3 expression. In fully differentiated adipocytes, SIRT3 overexpression activated macroautophagy, mainly on lipid droplets (LDs), through activating AMP-activated protein kinase (AMPK)-unc-51-like kinase 1 (ULK1) pathway, which in turn resulting in decreased LD size and lipid accumulation. Moreover, SIRT3 overexpression stimulated the formation of lysosome-associated membrane protein 2 (LAMP2)-heat shock cognate 71 kDa protein (HSC70)-perilipin-1 (PLN1) complex, to activate CMA and reduce the stability of LDs in adipocytes. In summary, the above results suggest that SIRT3 is a positive regulator of macroautophagy and CMA in adipocytes, which could be a potential therapeutic target for the treatment of obesity and its related metabolic disorders.