Objective To study the role of intestinal epithelial Tlr4 in systemic immune effects by establishing a mouse model of conditional Tlr4 knockout in intestinal epithelia. Methods Intestinal epithelial cell-specific Tlr4 gene knockout mice ( Tlr4^{f/ f} Cre T) were established by CRISPR/ Cas9 technology. The genotype of Tlr4^{f/ f} Cre T mice was identified by PCR and immunofluorescence. The general biological characteristics, reproductive capacity, and offspring survival rate were also investigated. Differences in immune organ structures, the intestinal mucosal immune cell ratio, and cytokine secretion levels between wildtype and Tlr4^{f/ f} Cre T mice were compared by HE staining, flow cytometry, and ELISAs. Results Establishment of Tlr4^{f/ f} Cre T mice was verified at gene and protein levels. Compared with wildtype mice, Tlr4^{f/ f} Cre T mice had no significant difference in general biological characteristics and offspring survival rate of > 90%. No significant differences in the physiological structure of the thymus, spleen, or liver, proliferation of splenic lymphocytes, and serum cytokine level, and intestinal mucosa were found between the two groups. However, the number of CD4+ T and γδT cells appeared to be significantly low in Tlr4^{f/ f} Cre T mice. Conclusions The intestinal epithelial cell- specific Tlr4 gene knockout mouse model was successfully established, which provides the experimental means to study the regulatory roles of Tlr4 expression of intestinal epithelial cells in diseases.