The ethanolic extract of I-Tiao Gung (GT-E) (Glycine tomentella root extract) was found to reduce the oxidative rate and prolonged lag phase of LDL in human (Homo sapiens) and tilapia (Oreochromis mossambicus). The in vivo effect of GT-E was determined using tilapia as a model. Hyperlipidemia and hypercholesterolemia were induced in fish by feeding commercial feed daily at 2% body mass for 8 weeks, or at 1% body mass for 12 weeks. Thirty two adult male tilapia were randomly divided into two groups and fed with feed containing 1% (w/w) GT-E or control diet for 12 weeks. Specific growth rate was similar between the GT-E group and the control group. Total triacylglycerol, total cholesterol and low-density lipoprotein cholesterol (LDL-C) in plasma of the GT-E group were significantly lower, while plasma total antioxidant status was significantly higher than those of the control group. GT-E fed fish had longer lag phase of Cu2+-induced LDL oxidation and retained more alpha-tocopherol in LDL particles than the control fish. LDL from the GT-E group had more monounsaturated fatty acids and less polyunsaturated fatty acids than the control group indicative of its effect on fatty acids metabolism. GT-E demonstrated hypolipidemic and hypocholesterolemic effects and inhibiting LDL oxidation in tilapia similar to the effects in mammals, thus tilapia can serve as a surrogate animal model for prescreening anti-atherosclerosis effect of natural products.