The effects of the turmeric ethyl acetate fraction (TEF) from the methanolic extract from Curcuma longa L. on lipid metabolism and underlying mechanisms of lipolysis were investigated in 3T3-L1 adipocytes. The intracellular lipid droplets were stained with Oil red O dye and quantified. Compared to the control, lipid accumulation was significantly decreased by 46.6% with treatment by TEF at the concentration of 20 microg/mL. The intracellular triglyceride (TG) level was also reduced by 37.9% at the concentration of 20 microg/mL. To determine the mechanism for TG content reduction, levels of glucose uptake and glycerol release were measured. Incubation of the 3T3-L1 adipocytes with TEF for 4 hours significantly lowered the cellular level of glucose in a dose-dependent manner. Furthermore, cellular expression of insulin-responsive glucose transporter (GLUT)-4 was decreased by 46%, indicating that reduced glucose uptake was due to a decrease in cellular GLUT-4 expression. In addition, the level of free glycerol released into the cultured medium was increased by 36.4% with the treatment by TEF. In subsequent measurements using quantitative real-time polymerase chain reaction, mRNA levels of hormone-sensitive lipase (HSL) and adipose TG lipase (ATGL) were elevated by 34.8% and 16.9%, respectively, at the concentration of 20 microg/mL. These results suggest that TEF partially inhibits lipogenesis by the suppression of glucose uptake via the decreased expression of cellular GLUT-4 and stimulates lipolysis through the induction of HSL and/or ATGL gene expression, resulting in the increased glycerol release.