Fatty acid synthase (FAS, E.C.2.3.1.85) played an essential role in fatty acid synthesis, and it was reported as a potential new therapeutic target for obesity recently; Tuber fleeceflower stem(Caulis polygoni multiflori) was a traditional Chinese medicine. Our study showed that tuber fleeceflower stem extract (TFS) could potently inhibit FAS. 40% ethanol behaved best in extracting FAS inhibitors from tuber fleeceflower stem. The half inhibition concentration (IC50) of TFS on FAS overall reaction was 0.61±0.024 g/ml, equivalent to dried herb weight of 3.48±0.14 g/ml. The inhibition kinetics revealed that the inhibition on FAS by TFS was non-competitive against substrate acetyl-CoA and malonyl-CoA, but it was more complicated against NADPH, potent inhibitors in TFS appeared to be various. Moreover, TFS could inhibit ketoacyl reduction of FAS with an IC50 value of 2.14±0.12 g/ml，which indicated that ketoacyl reductase in FAS was one of the reacted sites that TFS attacked. In addition, TFS significantly reduced food intake and body weight of diet-induced obesity SD rats and mice via oral administration, FAS activities in livers of the tested rats were lower than that of the control rats. These results would be very useful for the study of FAS inhibitors and the control of obesity.