Trans-fatty acid consumption from partially hydrogenated vegetable oil (PHVO) has been positively associated with multiple cardiovascular disease risk factors and events. This study was designed to examine the effects of trans-fatty acid isomer profile of PHVO on plasma lipids and lipoproteins and hepatic expression of key genes involved in cholesterol and fatty acid metabolism. Thirty-three male F(1)B strain Syrian Golden Hamsters were allocated to 1 of 3 hypercholesterolemic diets containing (5% by weight): 1) tristearin [control fat (CON)]; 2) partially hydrogenated high-oleic acid sunflower oil (PH-SUN); or 3) partially hydrogenated high-linoleic acid safflower oil (PH-SAF). PH-SUN contained more trans-4 to trans-10 18:1 compared with PH-SAF, which contained more trans-11 to trans-16 18:1. The addition of both PHVO to the diet increased plasma total cholesterol concentrations relative to CON, but only PH-SUN increased the plasma ratio of non-HDL:HDL cholesterol compared with CON. PH-SUN increased VLDL (total, large, and medium) and IDL particle concentrations while decreasing total, medium, and small HDL particle concentrations relative to CON. Both PHVO diets increased the hepatic cholesterol ester concentration, whereas the hepatic TG concentration was lower in PH-SUN compared with PH-SAF and CON. Levels of hepatic LDL receptor, HMG-CoA reductase, and sterol response element binding protein 1 mRNA were specifically reduced in the PH-SUN group compared to the CON group. Expression of SREBP1c was upregulated in both PHVO groups compared to CON, whereas only the PH-SAF group had higher levels of the lipogenic enzymes acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase-1 compared to CON. These results indicate that differences in the trans-fatty acid profile of PHVO can differentially affect lipid and lipoprotein metabolism.