Peroxisome proliferator-activated receptor-alpha (PPARalpha) is a major transcriptional regulator of lipid metabolism. It is activated by diverse chemicals such as fatty acids (FAs) and regulates the expression of numerous genes in organs displaying high FA catabolic rates, including the liver. The role of this nuclear receptor as a sensor of whole dietary fat intake has been inferred, mostly from high-fat diet studies. To delineate its function under low fat intake conditions (4.8% w/w), we studied the effects of five regimens with contrasted FA compositions on liver lipids and hepatic gene expression in wild-type and PPARalpha-deficient mice. Diets containing polyunsaturated FAs reduced hepatic fat stores in wild-type mice. Only sunflower, linseed, and fish oil diets lowered hepatic lipid stores in PPARalpha-/- mice, a model of progressive hepatic triglyceride accumulation. These beneficial effects were associated, in particular, with dietary regulation of Delta9-desaturase in both genotypes, and with a newly identified PPARalpha-dependent regulation of lipin. Furthermore, hepatic levels of 18-carbon essential FAs (C18:2omega6 and C18:3omega3) were elevated in PPARalpha-/- mice, possibly due to the observed reduction in expression of the Delta6-desaturase and of enoyl-coenzyme A isomerases. Effects of diet and genotype were also observed on the xenobiotic metabolism-related genes Cyp3a11 and CAR. CONCLUSION: Together, our results suggest that dietary FAs represent–even under low fat intake conditions–a beneficial strategy to reduce hepatic steatosis. Under such conditions, we established the role of PPARalpha as a dietary FA sensor and highlighted its importance in regulating hepatic FA content and composition.