Objective：This study aims to observe the effect of high fat diet on jejunal and ileal epithelial function in mice. Methods：A total of 40 male C57BL/6J mice aged 8 weeks were randomly divided into two groups and fed with normal chowdiet and high-fat diet respectively for 3 months. Jejunum and ileum tissues were collected from mice，and the morphological changes of villi and crypts in jejunum and ileum were detectedby H&E staining. PAS staining was used to detect the number of goblet cells. Crypt units were isolated for primary 3D organoid culture in vitro，and the percentage of organoid per crypt ratio and the number of crypt-domains per organoid were observed. The expression of apoptotic genes in jejunum and ileum were determined by PCR. Results：The villus length and goblet cell number of jejunum and ileum were not significantly affected by high fat diet，but the crypt depth was significantly shallower. The results of 3D organoid culture in vitro showed that the differentiation ability of jejunal epithelial stem cells decreased，but that of ileal stem cells increased significantly. And apoptosis of both kinds of epithelial cells was significantly reduced. Conclusion：High fat diet changed the function of jejunal and ileal epithelial cells in mice，and had different effects on the differentiation ability of jejunal and ileal epithelial stem cells.