The aim was to investigate if chronic feeding with oligofructose (OFS), a nondigestible fructan that decreases triacylglycerol-very-low-density lipoproteins (TAG-VLDLs) in the serum of rats by reducing hepatic de novo lipogenesis, could counteract the impact of fructose on TAG metabolism. Male Wistar rats fed a standard diet supplemented or not with 10% OFS for 30 days received either tap water or a 10% fructose drinking solution for 48 hours. TAG, phospholipids (PLs), cholesterol, and free fatty acids were assayed both in serum and in liver. Fatty acid de novo synthesis, esterification, and beta-oxidation were assessed in the liver by measuring the activity of key enzymes: fatty acid synthase (FAS), phosphatidate phosphohydrolase (PAP), glycerol-3-phosphate acyltransferase (GPAT), and carnitine palmitoyltransferase-I (CPT-I), respectively. The acute load of fructose increased (1) both liver and serum TAG without affecting other lipids, and (2) de novo fatty acid synthesis and esterification, through induction of FAS and PAP without affecting CPT-I. Long-term feeding with OFS protected rats against liver TAG accumulation induced by fructose. The lower lipogenic capacity of the liver could be the key event in this protection, since even after the fructose load FAS activity remained significantly lower in OFS-fed rats. However, despite its protective effect on the liver, OFS was not able to prevent fructose-induced hypertriglyceridemia, suggesting that OFS feeding could not counteract the fructose-induced defect in TAG-VLDL clearance.