We evaluated, in human cell line HepG2, the action of individual dietary polyunsaturated fatty acids (PUFAs) on the expression of several lipid metabolism genes. The effects of docosahexaenoic acid, 22:6, n-3 (DHA), eicosapentaenoic acid, 20:5, n-3 (EPA), and arachidonic acid, 20:4, n-6 (AA) were studied alone and with vitamin E (Vit.E). DHA, EPA, and AA down-regulated mRNAs and encoded proteins of stearoyl-CoA desaturase (SCD) and sterol regulatory element binding protein (SREBP-1c), two major factors involved in unsaturated fatty acids synthesis. DHA affected SREBP-1c mRNA less markedly than EPA and AA. Vit.E did not affect these products, both when individually added or together with fatty acids. The expression of UDP-glucuronosyl transferase 1A1 (UGT1A1) mRNA, an enzyme of phase II drug metabolism with relevant actions within lipid metabolism, resulted also differentially regulated. DHA did not essentially reduce UGT1A1 mRNA expression while EPA and AA produced a considerable decrease. Nevertheless, when these PUFAs were combined with Vit.E, which by itself did not produce any effect, the result was a reduction of UGT1A1 mRNA with DHA, an increase reverting to basal level with EPA and no variation with AA. Observed regulations did not result to be mediated by peroxisome proliferator-activated receptor (PPAR). Our data indicate that major dietary PUFAs and Vit.E are differentially and selectively able to affect the expression of genes involved in lipid metabolism. The different actions of these slightly different molecules could be associated with their physiological role as relevant nutrient molecules.