Members of the acyl-CoA thioesterase (Acot) gene family hydrolyze fatty acyl-CoAs, but their biological functions remain incompletely understood. Thioesterase superfamily member 2 (Them2; synonym Acot13) is enriched in oxidative tissues, associated with mitochondria, and relatively specific for long chain fatty acyl-CoA substrates. Using Them2(-/-) mice, we have demonstrated key roles for Them2 in regulating hepatic glucose and lipid metabolism. However, reduced body weights and decreased adiposity in Them2(-/-) mice observed despite increased food consumption were not well explained. To explore a role in thermogenesis, mice were exposed to ambient temperatures ranging from thermoneutrality (30 °C) to cold (4 °C). In response to short term (24-h) exposures to decreasing ambient temperatures, Them2(-/-) mice exhibited increased adaptive responses in physical activity, food consumption, and energy expenditure when compared with Them2(+/+) mice. By contrast, genotype-dependent differences were not observed in mice that were equilibrated (96 h) at each ambient temperature. In brown adipose tissue, the absence of Them2 was associated with reduced lipid droplets, alterations in the ultrastructure of mitochondria, and increased expression of thermogenic genes. Indicative of a direct regulatory role for Them2 in heat production, cultured primary brown adipocytes from Them2(-/-) mice exhibited increased norepinephrine-mediated triglyceride hydrolysis and increased rates of O2 consumption, together with elevated expression of thermogenic genes. At least in part by regulating intracellular fatty acid channeling, Them2 functions in brown adipose tissue to suppress adaptive increases in energy expenditure.