Hind limb-suspended rats represent a sedentary-hyperinsulinemic model with a liver dyslipidemia mainly related to changes in sterol regulatory element-binding protein 1 (SREBP-1) and peroxisome proliferator-activated receptor-α (PPARα) expression and activity. To assess the effects of dietary fatty acids on hepatic lipid homeostasis, the hepatic expression and activity of PPARα, SREBP-1, and hepatocyte nuclear factor-4α (HNF-4α) were investigated in this animal model. In control and sedentary rats, diets enriched with saturated, monounsaturated, and polyunsaturated fatty acids (PUFA) enhanced the expression of the PPARα target genes carnitine palmitoyltransferase 1 and acyl-CoA oxidase, the highest effect being exerted by ω-3. The same diets reduced SREBP-1 mRNA and target lipogenic gene expression, as indicated by the reduction in fatty acid synthase and acetyl-CoA carboxylase mRNA content. Effects were greater in sedentary rat liver than in controls on the same diet. Only the ω-3 enriched diet decreased liver triglyceride content as well as plasma cholesterol and triglyceride levels in sedentary rats. This effect may be mainly related to the enhanced mitochondrial and peroxisomal β-oxidation genes expression. On the other hand, saturated fatty acid-enriched diet induced an increase in liver triglyceride content and enhanced plasma cholesterol and triglyceride levels, both in control and immobilized rats. This detrimental effect may be ascribed to the induced HNF-4α binding activity on ApoCIII promoter and to the enhanced ApoCIII mRNA levels both in control and in sedentary rat livers. In conclusion, we can speculate that dietary saturated fats, acting at apolipoprotein transcriptional level, may impact on the close relationship existing among high ApoCIII plasma level, dyslipidemia, and atherosclerosis.