Dietary fatty acids modulate plasma and intracellular cholesterol concentrations. Circulating non-high-density lipoprotein cholesterol (nHDL-C) concentration is determined by rates of hepatic very low-density lipoprotein assembly and secretion, and clearance of subsequent metabolic products. The effect of dietary fat (butter, traditional margarine, soybean oil, and canola oil) was assessed with respect to plasma lipids, hepatic lipid composition, and messenger RNA (mRNA) abundance of low-density lipoprotein (LDL) receptor, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, sterol regulatory element-binding protein (SREBP) 2, and microsomal triglyceride transfer protein (MTP) in the Golden-Syrian hamster (Charles River Laboratories, Wilmington, MA). Hamsters were fed with a nonpurified diet (6.25 fat g/100 g) with 0.1 g cholesterol/100 g (control diet) or control diet with an additional 10 g experimental fat/100 g for 12 weeks. Hamsters fed with the control diet, unsaturated fats (canola and soybean oils), and margarine, relative to butter, had significantly lower total cholesterol and nHDL-C and triglyceride concentrations. Additional dietary fat, regardless of fatty acid profile, resulted in higher hepatic cholesterol concentrations. In contrast, relative to the control diet-, butter-, or margarine-fed hamsters, these changes were associated with a 4- and 8-fold higher LDL receptor and 5- and 9-fold higher SREBP mRNA abundance, in hamsters fed with canola and soybean oils, respectively. MTP mRNA, a marker of very low-density lipoprotein particle formation, was higher in canola- and soybean oil-fed hamsters relative to the control diet-fed hamsters, although differences were modest. These results suggest that the substitution of canola and soybean oils for butter results in lower nHDL-C concentrations that may be related to increased mRNA abundance of the LDL receptor, SREBP-2, and MTP genes.