Recent advancements suggest that in addition to its roles in developmental processes, transcription repressor hairy and enhancer of split 1 (Hes1) also acts as a key regulator of inflammatory responses. A healthy gut microbiota ecology is critical for establishment of tissue homeostasis. However, the role of epithelial Hes1 in regulating intestinal microbiota ecology and intestinal homeostasis remains unexplored. Here we show that epithelial Hes1 deficiency leads to intestinal microbial dysbiosis and disturbed homeostasis. Both inducible Hes1 deletion and intestinal epithelial cell (IEC)-intrinsic Hes1 deletion resulted in loss of Bacteroidetes in ileum and increase of Escherichia coli and Akkermansia muciniphila in colon. Loss of Bacteroidetes closely correlated with decreased expression of commensal-dependent antimicrobial genes, leading to impaired resistance against pathogenic bacterial colonization. Moreover, Hes1 deficiency enhanced susceptibility to Dextran sodium sulphate-induced intestinal inflammation. Of note, transfer of Hes1-deficient-mouse-derived fecal microbiota promoted intestinal inflammation. The increase of A. muciniphila in colon was associated with Hes1-deficiency-induced unbalanced mucosal microhabitats. Thus, our results support that IEC-intrinsic Hes1 maintains gut homeostasis by preventing microbial dysbiosis partially through regulating mucosal microhabitats.