BACKGROUND: Owing to the distinctive advantages of mesenchymal stem cell-derived exosomes (MSCs-exo), these vesicles have emerged as a pivotal research focus in regenerative medicine, surpassing their MSC counterparts. Quercetin (Qr), widely recognized for its potent anti-inflammatory and antioxidant activities, demonstrates substantial potential in enhancing tissue repair processes. This study delves into the role of quercetin-pretreated MSC-derived exosomes (MSCs-exo) in accelerating the healing of diabetic wounds. METHODS: MSCs-exo were isolated from quercetin-pretreated MSCs and applied to fibroblasts to evaluate changes in cell function. An in vitro DSW rat model was also developed, and the rats were treated with MSCs-exo to assess wound healing progression. Fecal samples were collected for 16S rRNA sequencing and untargeted metabolomics to analyze changes in gut microbiota and metabolic profiles. RESULTS: MSCs-exo significantly enhanced fibroblast proliferation and migration while improving the therapeutic efficacy of MSCs-exo in DSW treatment. Gut microbiota and metabolomic analyses revealed marked changes in DSW rats, with MSCs-exo effectively alleviating dysbiosis. MSCs-exo upregulated Faecalibacterium abundance and regulated arachidonic acid metabolism in both the arachidonic and linoleic acid pathways. Firmicutes and Enterobacteriaceae influenced the arachidonic acid pathway by modulating 14.15-EET expression levels. CONCLUSION: MSCs-exo facilitate DSW wound healing through modulation of dysbiotic gut microbiota linked to DSW pathology. This discovery offers novel therapeutic avenues and research trajectories for enhancing DSW recovery.