Long-term high-fat feeding (HF) induces intestinal mucosal barrier damage. However, the mechanism for this remains unclear. HF can elevate the intestinal and circulating bile acid (BA) levels, especially deoxycholic acid (DCA). We hypothesize that BAs elevated by HF regulate intestinal stem cell (ISC) function, which may contribute to mucosal barrier injury in the ileum of mice. In this study, we showed that 2 weeks of HF resulted in a shortening of intestinal villi and a decrease in the tight junction (TJ) protein occludin in the ileum of mice, accompanied by an increase in circulating BA levels. Importantly, 2 weeks of HF also reduced ileal ISCs and goblet cells and decreased the proliferation function of ISCs and their ability to differentiate into goblet cells. Endoplasmic reticulum (ER) stress was found to be involved in the process of ISC damage. All these alterations were reversed by cofeeding with the bile acid binder cholestyramine. In addition, the in vitro studies also confirmed a cytotoxic effect of DCA at a high concentration on ISCs and goblet cells. In conclusion, these data suggested that high levels of BAs induced by HF could impair ISC function by triggering ER stress, resulting in the disruption of the intestinal mucosal barrier. (C) 2020 Elsevier Inc. All rights reserved.