Obesity is characterized by a low-grade inflammation associated with disturbances of small intestine permeability. The latter notably relies on mitochondrial function of intestinal epithelial cells (IEC). Yet changes in lipid metabolism of IEC induced by high fat diet (HFD) might alter mitochondrial function of IEC. We thus wondered whether saturated fats alter mitochondrial function of IEC and intestinal permeability. The in vitro model of enterocyte IPEC-J2 was treated for 3 days with lauric (C12:0), myristic (C14:0), palmitic (C16:0) and stearic (C18:0) acids, abundantly found in HFDs, alone at 250 μM or in mix with 250 μM each. Mitochondrial function was assessed by the Seahorse technology while epithelial permeability was evaluated by measuring the transepithelial electrical resistance. Treatment with the mix of fatty acids induced enterocyte steatosis and oxidative stress concomitant to antioxidant machinery activation but decreased β-oxidation activity during the first hours of treatment, indicating enterocyte metabolic adaptation. Mitochondrial function was altered after 3 days of treatment with marked decreased respiration and lower mitochondrial ATP production rate linked with increased epithelial permeability compared to control cells. Although 3 days of treatment with each fatty acid provoked enterocyte steatosis, C12:0 and C14:0 induced greater lipid storage than C16:0 and C18:0. Only C16:0 decreased mitochondrial respiration of IPEC-J2 while C16:0 and C18:0 lowered the mitochondrial ATP production rate and increased epithelial permeability. In conclusion, chronic treatment with a mix of fatty acids simulating HFD, induced alterations of enterocyte metabolism and increased permeability likely due to the effect of C16:0 and/or C18:0.