Obesity and its related disorders have been associated to the presence in the blood of gut bacteria-derived lipopolysaccharides (LPS). However, the factors underlying this low-grade elevation in plasma LPS, so-called metabolic endotoxemia, are not fully elucidated. We aimed to investigate the effects of Western diet (WD) feeding on intestinal and hepatic LPS handling mechanisms in a rat model of diet-induced obesity (DIO). Rats were fed either a standard chow diet (C) or a Western Diet (WD, 45% fat) for 6 weeks. They were either fed ad libitum or pair-fed to match the caloric intake of Crats for the first week then fed ad libitum for the remaining 5 weeks. Six-week WD feeding led to a mild obese phenotype with increased adiposity and elevated serum LPS-binding protein (LBP) levels relative to C rats, irrespective of initial energy intake. Serum LPS was not different between dietary groups but exhibited strong variability. Disrupted ileal mucus secretion and decreased ileal Reg3- and -β gene expression along with high ileal permeability to LPS were observed in WD compared to C-fed rats. Ileal and caecal intestinal alkaline phosphatase (IAP) activity as well as Verrucomicrobia and Bifidobacterium caecal levels were increased in WD-fed rats compared to C-fed rats. WD consumption did not impact mRNA levels of LPS-handling hepatic enzymes. Correlation analysis revealed that ileal passage of LPS, IAP activity, Proteobacteria levels and hepatic aoah gene expression correlated with serum LPS and LBP, suggesting that ileal mucosal defense impairment induced by WD feeding contribute to metabolic endotoxemia.