The effects of Aster scaber seed oil (ASO) on lipid profiles were studied in rats and hamsters. ASO contained considerable amounts of Δ3t-16:1 (11.4%), Δ3t, 9c-18:2 (4.6%), and Δ3t, 9c, 12c-18:3 (11.3%). Young rats and hamsters were fed diets containing ASO, soybean oil (SBO), or olive oil (OLO) as fat sources for 4 weeks in separate experiments with or without cholesterol. In the rat study, there were no significant differences in the concentrations of serum total cholesterol, high-density lipoprotein (HDL)-cholesterol, and triacylglycerol among the groups. The serum but not liver malondialdehyde (MDA) level was significantly lower in the ASO-fed group than it was in the other groups. The biochemical and growth parameters revealed no significant biological damages in the ASO-fed animals. In the hamster study, dietary cholesterol-dependent effects were evident in the serum lipids profiles, whereas the fat-induced effect was only observed in the ratio of serum low-density lipoprotein (LDL)-/HDL-cholesterol. Furthermore, fat- and cholesterol-induced effects were evident in the ratio of serum LDL-/HDL-cholesterol. Significant interactions between dietary fat and cholesterol were observed as evident from the concentration of serum cholesterol and triacylglycerol, as well as the activity of serum cholesterol ester transfer protein. These results suggest that dietary ASO containing trans-Δ3 fatty acids appeared to improve the serum LDL-/HDL-cholesterol ratio more than the SBO did, especially when hamsters were simultaneously fed cholesterol-supplemented diet.