Although stem cell transplantation is an effective strategy in the treatment of type 1 diabetes mellitus (T1DM), the mechanisms underlying its therapeutic effects remain unclear. We hypothesized that stem cells target gut microbiota and intestinal mucosal immunity to promote therapeutic effects against T1DM. We investigated the effects of human amniotic mesenchymal stem cells (hAMSCs) on intestinal microbiota and mucosal immunity in streptozotocin-induced T1DM mice. hAMSCs promoted significant reductions in blood glucose levels and increased the number of insulin-secreting cells in the T1DM model. Compared with T1DM model mice, 16S rRNA sequencing revealed significant differences in the composition, diversity, and abundance of microbiota in the ileum of hAMSC-treated mice. Bifidobacterium, Prevotella, and Alcaligenes species were among the 15 most abundant differential bacterial species. LC-MS revealed significant changes in ileal metabolites, and among the top 100 differential metabolites identified, we found that a significant increase in taurine was closely associated with hAMSC therapy. Additionally, we detected significant differences between the two groups with respect to the frequency and phenotype of CD4⁺ T cell subsets in mesenteric lymph nodes, and hAMSCs promoted significant increases in Th2 and Treg cell frequencies and reduced the frequencies of Th1 and Th17 cells. Moreover, correlation analysis revealed pairwise correlations between differential microflora and differential metabolites and immune signatures. hAMSCs thus have positive effects on the microbiota and their metabolites in the ileum and intestinal mucosal immunity in T1DM. Our findings indicate that gut microbiota and intestinal mucosal immunity may play vital roles in the hAMSC-based treatment of T1DM.