Increased luminal bile acid hydrophobicity is associated with cytotoxicity and has been suggested to contribute to gut barrier dysfunction. The aim of this study was to compare 2 high-fat diets and a low-fat diet as to whether they modify fecal bile acid profile and hydrophobicity and/or susceptibility to dextran sodium sulfate (DSS) colitis in C57Bl/6J mice. Control and DSS-Control groups received a low-fat control diet [5.5% of total energy (E%) soy oil, 4.5 E% lard], and the DSS-Lard (5.5 E% soy oil, 54.5 E% lard) and DSS-Fish oil (5.5 E% soy oil, 27.2 E% lard and 27.2% menhaden oil) groups received high-fat diets. Feces for bile acid analysis were collected after 3-wk feeding, followed by induction of dextran DSS colitis (2 d 5% DSS in drinking water + 2 d tap water). Fecal bile acid hydrophobicity was elevated 76% in the lard group (P = 0.051) and 122% in the fish oil group (P = 0.001) compared with control, indicating potentially increased cytotoxicity. DSS caused severe colitis symptoms, evaluated as rectal bleeding, whereas all the controls were symptom free. The median symptom scores were: DSS-Control, 2.3 (IQR = 0.6, 3.0); DSS-Lard, 0.3 (IQR = 0, 2.3); and DSS-Fish oil, 2.4 (IQR = 1.9, 2.8). The only differences were DSS-Control vs. control (P < 0.001) and DSS-Fish oil vs. control (P < 0.001). Severity of symptoms in all colitic mice was positively correlated with fecal bile acid hydrophobicity (Spearman's ρ = 0.43; P = 0.028) and fecal deoxycholic acid concentration (Spearman's ρ = 0.39; P = 0.048). These results suggest that luminal bile acid modification, induced by altered dietary fat composition, may alter susceptibility to DSS colitis.