Thylakoids reduce body weight gain and body fat accumulation in rodents. This study investigated whether an enhanced oxidation of dietary fat-derived fatty acids in the intestine contributes to the thylakoid effects. Male Sprague-Dawley rats were fed a high-fat diet with (n = 8) or without thylakoids (n = 8) for two weeks. Body weight, food intake and body fat were measured, and intestinal mucosa was collected and analyzed. Quantitative real-time polymerase chain reaction (q-RT-PCR) was used to measure gene expression levels of key enzymes involved in fatty acid transport, fatty acid oxidation and ketogenesis. Another set of thylakoid-treated (n = 10) and control rats (n = 10) went through indirect calorimetry. In the first experiment, thylakoid-treated rats (n = 8) accumulated 25% less visceral fat than controls. Furthermore, fatty acid translocase (Fat/Cd36), carnitine palmitoyltransferase 1a (Cpt1a) and mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2) genes were upregulated in the jejunum of the thylakoid-treated group. In the second experiment, thylakoid-treated rats (n = 10) gained 17.5% less weight compared to controls and their respiratory quotient was lower, 0.86 compared to 0.91. Thylakoid-intake resulted in decreased food intake and did not cause steatorrhea. These results suggest that thylakoids stimulated intestinal fatty acid oxidation and ketogenesis resulting in an increased ability of the intestine to handle dietary fat. The increased fatty acid oxidation and the resulting reduction in food intake may contribute to the reduced fat accumulation in thylakoid-treated animals.