Objective To construct and identify macrophage-conditional Cd226 gene knockout mice, and to provide an animal model for studying the effect of CD226 on the occurrence and development of diseases by regulating the phenotype and function of macrophages. Methods Cd226^{flox/ +} mice were self-crossed by male and female to obtain Cd226^{flox/ flox} mice. Cd226^{flox/ flox} were hybridized with Lyz2-Cre⁺ mice to obtain Cd226^{flox/ +}Lyz2-Cre⁺ mice, and then crossed with Cd226^{flox/ flox} mice to obtain macrophage-conditional Cd226 knockout mice(Cd226^{flox/ flox} Lyz2-Cre⁺ ). The phenotypes of the mice were identified by PCR and agarose gel electrophoresis. To verify that CD226 had been conditionally knockdown in macrophages, qRT-PCR, flow cytometry and Western Blot were used to evaluate CD226 expression. Transwell was used to dectect the effect of CD226 on the migration of macrophages. Results The successful establishment of macrophage-conditional Cd226 gene knockout mice was confirmed at gene and protein levels. Compared with Cd226^{flox/ flox} mice, the migration ability of peritoneal macrophages was significantly inhibited in Cd226^{flox/ flox} Lyz2-Cre⁺ mice. Conclusions Macrophage-conditional Cd226 gene knockout mice were successfully established, which can provide a more accurate animal model for studying the role and mechanism of CD226 regulation of macrophage in the pathogenesis of immune diseases.