Objective To investigate the immunoregulatory effects of CD226 on the chronic restraint stress (CRS)-induced depression-like behavior and its underlying mechanism in mice. Methods Male C57/BL6J mice and CD226 gene knockout (KO) mice with the same strain (4-6 week old) were adopted to establish CRS models. The stress-induced depression scores of mice were evaluated through behavioral testing such as forced swimming test and sucrose preference test. Flow cytometry was used to analyze the differences of the intraepithelial lymphocytes in the spleens, peyer's patches, and intestines between the two groups. Results Compared with WT CRS group, mice in CD226KO CRS group showed significantly decreased immobility time in forced swimming test and increased sucrose preference rate. The ratio of CD4+ T cells to CD8+ T cells in spleen was significantly reduced, combined with the remarkably elevated proportion of TCRαβ and TCRαβCD8αβ cells in the small intestinal IELs of CD226 KO mice with CRS. Conclusion Knockout of CD226 alleviates CRS-induced depression-like behavior in mice, alters the proportion of immune cells in murine spleen and intestine, and improves the overall immune status of mice under stress.