Intestinal mucosal immunity plays a pivotal role in host defence. In this study, we found that cluster of differentiation 226 (CD226) gene knockout (KO) led to more severe atopic dermatitis (AD)‐related skin pathologies and bowel abnormalities in a 2,4‐dinitrochlorobenzene (DNCB)‐induced AD‐like mouse model. Following DNCB administration, the expression of CD226 was elevated in intestinal mucosal tissues, including group 3 innate lymphoid cells (ILC3s) and CD4+ T cells of Peyer's patches (PPs). CD226 deficiency led to an overactive intestinal immune response in the AD‐like mice, as evidenced by increased inflammation and Th1/Th2‐related cytokine levels as well as increased Paneth cell numbers and antimicrobial peptide (AMP) expression, which was likely due to the higher interleukin (IL)‐22 production in the lamina propria. Additionally, CD226 deficiency increased the production of IL‐4 and IL‐17 in mesenteric lymph nodes as well as the number of PPs and expression of immunoglobulin (Ig) A in B cells. Moreover, insufficient expression of CD226 affected the characterization of intraepithelial and lamina propria lymphocytes in the intestinal mucosa. Finally, the number of PPs was increased in CD4+ T cell‐specific CD226 KO and regulatory T (Treg) cell‐specific CD226 KO mice; thus, loss of CD226 in Treg cells resulted in impaired Treg cell‐suppressive function. Therefore, our findings indicate that CD226 deficiency alters intestinal immune functionality in inflammatory diseases.