Objective To investigate the effects of colonic epithelial estrogen receptor β（ERβ） deletion on colonic tryptophan hydroxylase-1（TPH1） and 5-hydroxytryptamine（5-HT） levels in C57BL/6J mice. Methods Pvillin-Cre^+/- mice and ERβflox^+/- mice were obtained using CRISPR-Cas9 technology，and intestinal ERβ knockout homozygous（ERβflox^-/--Pvillin-Cre^+/+，ERβCKO，n=5），heterozygous（ERβflox^+/--Pvillin-Cre^+/+，ERβCKO^+/-n=5），and littermate wild-type（WT） mice were ultimately obtained by mating and breeding. Mice of each strain at 6 and 12 months of age were used for the study. Hematoxylin-eosin staining（HE），enzyme-linked immunosorbent assay（ELISA），Western blotting（WB），and immunohistochemistry（IHC） were used for determining the intestinal morphology and measuring intestinal ERβ，5-HT，and TPH1 levels of mice in each group. Results After the successful construction of intestinal ERβCKO mice as verified by gene identification，WB showed that intestinal ERβ levels were significantly down-regulated（P<0.05）. HE showed abnormalities in intestinal structural integrity and intestinal mucosal epithelial villi development after intestinal ERβ knockdown. ELISA results showed that intestinal 5-HT levels of ERβCKO mice at 6 and 12 months of age were significantly decreased compared with those of WT mice（P<0.05），and the decrease was in a month-age-dependent manner. IHC results showed that TPH1 was expressed in the intestines of mice of all groups. However，ERβCKO mice showed a reduced number of TPH1 immunoreactive cells（P<0.05） and lower expression of immunoreactive substances（P<0.05） compared with WT mice，but the decline in TPH1 was not in a month-age-dependent manner. Conclusion Down-regulation of colonic ERβ induces a decrease in colonic TPH1 protein and 5-HT levels，which subsequently triggers abnormal intestinal function and increases the risk of developing intestinal diseases in mice. This study is expected to provide a laboratory basis for the pathogenesis of ERβ-targeted intestinal diseases.