Adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triacylglycerol (TG) hydrolysis, is long known to be a phosphoprotein. However, the potential phosphorylation events that are involved in the regulation of ATGL function remain incompletely defined. Here, using a combinatorial proteomics approach, we obtained evidence that at least eight different sites of ATGL can be phosphorylated in adipocytes. Among them, Thr-372 resides within the hydrophobic region known to mediate lipid droplet (LD) targeting. While it had no impact on the TG hydrolase activity, substitution of phosphorylation-mimic Asp for Thr-372 eliminated LD localization and LD-degrading capacity of ATGL expressed in HeLa cells. In contrast, mutation of Thr-372 to Ala gave a protein that bound LDs and functioned the same as the wild type protein. In non-stimulated adipocytes, the Asp mutation led to decreased LD association and basal lipolytic activity of ATGL while the Ala mutation produced opposite effects. Moreover, the LD translocation of ATGL upon β-adrenergic stimulation was also compromised by the Asp mutation. In accordance, the Ala mutation promoted and the Asp mutation attenuated the capacity of ATGL to mediate lipolysis in adipocytes under both basal and stimulated conditions. Collectively, these studies identified Thr-372 as a novel phosphorylation site that may play a critical role in determining subcellular distribution as well as lipolytic action of ATGL.