Interesterified fats have largely replaced hydrogenated vegetable fat, which is rich in trans fatty acids, in the food industry as an economically viable alternative, generating interest to study their health effects. The aim of this study was to evaluate the effect that interesterification of oils and fat has on lipid-induced metabolic dysfunction, hepatic inflammation and ER stress. Five week-old male Wistar rats were randomly divided into three experimental groups, submitted to either normocaloric and normolipidic diet containing 10% of lipids from unmodified soybean oil (SO) or from interesterified soybean oil (ISO), and one more group submitted to a high fat diet (HFD) containing 60% of fat from lard as a positive control, for 8 or 16 weeks. Metabolic parameters and hepatic gene expression were evaluated. The HFD consumption led to increased body mass, adiposity and impaired glucose tolerance compared to SO and ISO at both time points of diet. However, the ISO group showed an increased body mass gain, retroperitoneal WAT mass, fasting glucose, and impaired glucose tolerance during ipGTT at 16 weeks compared to SO. Moreover, at 8 weeks, hepatic gene expression of Atf3 and Tnf were increased in the ISO group compared to the SO group. Thus, replacement of natural fat with interesterified fat on a normocaloric and normolipidic diet negatively modulated metabolic parameters and resulted in impaired glucose tolerance in rats.