We investigated mechanisms whereby PPAR agonism affects glucose and lipid metabolism in brown adipose tissue (BAT) by studying the impact of PPAR activation on BAT glucose uptake and metabolism, lipogenesis and mRNA levels plus activities of enzymes involved in triacylglycerol (TAG) synthesis. Interscapular BAT of rats treated or not with rosiglitazone (15 mg/kg/day, 7 days) was evaluated in vivo for glucose uptake and lipogenesis and in vitro for glucose metabolism, gene expression and activities of glycerolphosphate acyltransferase (GPAT), phosphatidate phosphatase-1 (PAP or lipin-1) and diacylglycerol acyltransferase (DGAT). Rosiglitazone increased BAT mass without affecting whole tissue glucose uptake. BAT glycogen content (-80%), its synthesis from glucose (-50%), and mRNA levels of UDP-glucose pyrophosphorylase (UDPG-PPL, -40%), which generates UDP-linked glucose for glycogen synthesis, were all reduced by rosiglitazone. In contrast, BAT TAG-glycerol synthesis in vivo and glucose incorporation into TAG-glycerol in vitro were stimulated by the agonist along with the activities and mRNA levels of glycerol 3-phosphate-generating phosphoenolpyruvate carboxykinase (PEPCK) and glycerokinase (GyK). Furthermore, rosiglitazone markedly increased the activities of glycerol 3-phosphate acyltransferase (GPAT) and diacylglycerol acyltransferase (DGAT), but not that of lipin1-mediated phosphatidate phosphatase-1, enzymes involved in the sequential acylation of glycerol 3-phosphate and TAG synthesis. Because an adequate supply of fatty acids is essential for BAT non-shivering thermogenesis, the enhanced ability of BAT to synthesize TAG under PPAR activation may constitute an important mechanism by which lipid substrates are stored in preparation for an eventual thermogenic activation.