This study assessed the effects of individual conjugated linoleic acid isomers, c9t11-CLA and t10c12-CLA, on nonalcoholic fatty liver disease (NAFLD) and systemic endothelial dysfunction in rats fed for four weeks with control or high-fructose diet. The high-fructose diet hampered body weight gain (without influencing food intake), increased liver weight and glycogen storage in hepatocytes, upregulated expression of fatty acid synthase (FAS) and stearoyl-CoA desaturase-1 (SCD-1), and increased saturated fatty acid (SFA) content in the liver. Both CLA isomers prevented excessive accumulation of glycogen in the liver. Specifically, t10c12-CLA decreased concentration of serum triacylglycerols and LDL + VLDL cholesterol, increased HDL cholesterol, and affected liver lipid content and fatty acid composition by downregulation of liver SCD-1 and FAS expression. In turn, the c9t11-CLA decreased LDL+VLDL cholesterol in the control group and downregulated liver expression of FAS without significant effects on liver weight, lipid content, and fatty acid composition. In summary, feeding rats with a high-fructose diet resulted in increased liver glycogen storage, indicating the induction of gluconeogenesis despite simultaneous upregulation of genes involved in de novo lipogenesis. Although both CLA isomers (c9t11 and t10c12) display hepatoprotective activity, the hypolipemic action of the t10c12-CLA isomer proved to be more pronounced than that of c9t11-CLA.