Mesenchymal stem cells (MSCs) therapy is a potential treatment strategy for ulcerative colitis (UC). The expression of the Id2 (inhibitor of differentiation factor-2) gene is closely associated with the pathogenesis and prognosis of UC. However, the role of Id2 in the therapeutic efficacy of MSCs for UC remains unclear. In this study, the MSCs that either overexpressed or knocked down the Id2 gene were employed to alleviate dextrose sodium sulfate (DSS) induced UC in mice. The results indicated that MSCs overexpressing Id2 showed no significant therapeutic advantage in MSCs for UC treatment. In contrast, MSCs with Id2 knockdown demonstrated a marked reduction in therapeutic efficacy on UC, evidenced by decreased body weight, elevated disease activity index (DAI), shortened colon length, increased histopathological damage, disruption of the colonic mucosal barrier, elevated levels of pro-inflammatory cytokines, and reduced tuft cell densities in mice. Notably, Id2 knockdown in MSCs impaired their ability to regulate intestinal microbiota in UC mice, significantly promoting the growth of potentially pathogenic bacteria such as Oscillibacter and Escherichia-Shigella, while decreasing the abundance of anti-inflammatory bacteria like Dubosiella. Transcriptome sequencing analysis revealed altered gene expression involved in immune regulation and signaling pathways (Wnt and Notch), including downregulation of CD1D, CD83, SAMHD1, PRRX1, AXIN2, JAG1, and DLL1, alongside upregulation of SPHK1. Our findings underscore the pivotal role of Id2 in the therapeutic efficacy of MSC treatments for UC.