Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme for triacylglycerol (TAG) hydrolysis in adipocytes. The precise mechanisms whereby ATGL is regulated remain uncertain. Here, we demonstrate that a protein encoded by G(0)/G(1) switch gene 2 (G0S2) is a selective regulator of ATGL. G0S2 is highly expressed in adipose tissue and differentiated adipocytes. When overexpressed in HeLa cells, G0S2 localizes to lipid droplets and prevents their degradation mediated by ATGL. Moreover, G0S2 specifically interacts with ATGL through the hydrophobic domain of G0S2 and the patatin-like domain of ATGL. More importantly, interaction with G0S2 inhibits ATGL TAG hydrolase activity. Knockdown of endogenous G0S2 accelerates basal and stimulated lipolysis in adipocytes, whereas overexpression of G0S2 diminishes the rate of lipolysis in both adipocytes and adipose tissue explants. Thus, G0S2 functions to attenuate ATGL action both in vitro and in vivo and by this mechanism regulates TAG hydrolysis.