The mechanism of adipose tissue lipolysis has not been fully elucidated. Greater understanding of this process could allow for increased feed efficiency and reduced fat in poultry. Studies in avian species may provide important insight in developing therapies for human obesity, as lipolytic pathways are highly conserved. Adipose triglyceride lipase (ATGL) cleaves triacylglycols, releasing non-esterified fatty acids (NEFA) into the bloodstream. Glucocorticoids have been shown to elevate circulating NEFA. To determine the regulation of ATGL and regulator proteins comparative gene identification-58 (CGI-58) and G(0)/G(1) switch gene 2 (G0S2) by glucocorticoid, 36 chickens received an injection of dexamethasone (4 mg/kg). Saline was administered to an additional 12 birds to determine any effect of stress during handling. Dexamethasone-injected birds were harvested at 0, 0.5, 1, 2, 4, and 6 h after treatment; saline-treated birds were collected at 4 and 6 h. Abdominal and subcutaneous adipose tissue and blood were collected. Gene and protein expression were analyzed via quantitative real-time PCR and western blot. Compared with the saline group, ATGL expression increased in birds injected with dexamethasone. When dexamethasone response was compared to the untreated group up to 6 h following injection, an increase in ATGL protein was observed as quickly as 0.5 h and increased further from 1 to 6 h. Plasma NEFA and glucose increased gradually from 0 to 6 h, reaching statistical significance at 4 h. These data show that ATGL expression is stimulated by glucocorticoid in a time-dependent manner.