Dietary supplementation with oligofructose (OFS; 100 g/kg), a non-digestible oligomer of beta-D-fructose, decreases serum triacylglycerols in serum and VLDL of rats. In order to investigate the role of hepatic metabolism in the hypolipidaemic effect of OFS, male Wistar rats were fed on a standard diet with or without 100 g Raftilose P95/kg as OFS source for 30 d. OFS feeding (1) significantly decreased triacylglycerol and phospholipid concentrations in both blood and liver, (2) increased the glycerol-3-phosphate liver content but decreased the hepatic activity of glycerol-3-phosphate acyltransferase (EC 2.3.1.15), suggesting a decrease in acylglycerol synthesis, (3) did not affect the blood non-esterified fatty acid concentrations, but (4) reduced by 54% the capacity of isolated hepatocytes to synthesize and secrete triacylglycerols from labelled acetate; the activity of fatty acid synthase, a key lipogenic enzyme was also significantly decreased. These findings suggest that OFS decreases serum triacylglycerols by reducing de novo fatty acid synthesis in the liver; the lower insulin level in the serum of OFS-fed rats could explain, at least partly, the metabolic effect induced by such non-digestible carbohydrates.