Foods contain bioactive components that contribute to optimal health. Food-grade yeast may contain components that enhance cellular glucose metabolism. We tested the effect of brewer's yeast (Saccharomyces cerevisiae) extract (YE), in vitro on rat fat cell glucose transport, glucose metabolism to lipid, and lipolysis. YE was fractionated by reverse-phase chromatography on a C18 open column using ammonium acetate (0.05 mol/L, pH 5.8), with acetonitrile (40%) elution solvent into fraction 1 (Fx1), fraction 2 (Fx2) and fraction 3 (Fx3). Isolated rat adipocytes were preincubated with insulin (51 pmol/L), YE (10 mg/L) or both; transport of U-(14)C-glucose was measured. Adipocytes were incubated with insulin and YE fractions (10 mg/L); glucose metabolism to lipid was measured by incorporation of U-(14)C-glucose into total lipids. Lipolysis was measured by glycerol release. Insulin stimulated glucose transport to sevenfold the basal value (P < 0.05). YE did not affect glucose transport. Insulin stimulated glucose metabolism to 2.6-fold the basal value (P < 0.001); YE stimulated glucose metabolism 14% (P < 0.005). YE potentiated the action of insulin 30% (P < 0.002). YE Fx2 and Fx3 stimulated glucose metabolism 25-40% (P < 0.05). Insulin inhibited lipolysis 47% (P < 0.001). YE alone inhibited lipolysis 63% (P < 0.001). YE and insulin inhibited lipolysis 81% (P < 0.001). Fractions of YE inhibited lipolysis in the presence of insulin (P < 0.05); the order of potency was Fx2 = Fx3 >> Fx1. A novel yeast extract (YE) and its fractions affect pathways of adipocyte metabolism differentially. YE and its fractions are good candidates for in vivo study.