The purpose of this study was to investigate the relationship between the lipid effects, the phenolic content, and the antioxidative effects of an ethyl acetate extract of fenugreek. Wistar rats fed a standard laboratory diet or cholesterol-rich diets for 16 weeks were used. The plasma lipid levels, total phenolics, and total flavonoid contents were measured, and the thiobarbituric acid reactive substances (TBARS) and antioxidant activities were examined. Administration of fenugreek ethyl acetate extract significantly lowered the plasma levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C), while increasing the plasma level of high-density lipoprotein cholesterol (HDL-C). Furthermore, the content of TBARS and catalase (CAT) and superoxide dismutase (SOD) activities in liver, heart and kidney decreased significantly after oral administration of the extract compared with those of rats fed a cholesterol-rich diet. These lipid effects and in vivo antioxidative effects were correlated with the in vitro phenolic content scavenging ability. In addition, three flavonoids (kaempferol 3-O-glycoside, apigenin-7-O-rutinoside, and naringenin) were identified by using an LC-MS/MS apparatus. Naringenin was the abundant flavonoid compound in the ethyl acetate extract, and its concentration reaches 7.23 +/- 0.09 mg/g of dry extract. These results revealed significant hypocholesterolemic effects and antioxidant activity in an ethyl acetate extract of fenugreek seed, which may be partly due to the presence of flavonoids, especially naringenin.