Abstract Objectives: This study aimed to explore the effects of bone marrow-derived Mesenchymal Stem Cell-Conditioned Medium (MSC-CM) treating diabetic foot ulcers in rats. Methods: Models of T2DM rats were induced by a high-fat diet and intraperitoneal injection of STZ in SD rats. Models of Diabetic Foot Ulcers (DFUs) were made by operation on hind limbs in diabetic rats. Rats were divided into four groups (n = 6 for each group), i.e., Normal Control group (NC), Diabetes Control group (DM-C), MSC-CM group and Mesenchymal Stem Cells group (MSCs). MSC-CM group was treated with an injection of conditioned medium derived from preconditioned rats’ bone marrow MSCs around ulcers. MSCs group were treated with an injection of rats’ bone marrow MSCs. The other two groups were treated with an injection of PBS. After the treatment, wound closure, re-epithelialization (thickness of the stratum granulosums of the skin, by H&E staining), cell proliferation (Ki67, by IHC), angiogenesis (CD31, by IFC), autophagy (LC3B, by IFC and WB; autoly-sosome, by EM) and pyroptosis (IL-1β, NLRP3, Caspase-1, GSDMD and GSDMD-N, by WB) in ulcers were evaluated. Results: After the treatment wound area rate, IL-1β by ELISA, and IL-1β, Caspase-1, GSDMD and GSDMD-N by WB of MSC-CM group were less than those of DM group. The thickness of the stratum granulosums of the skin, proliferation index of Ki67, mean optic density of CD31 and LC3B by IFC, and LC3B by WB of MSC-CM group were more than those of DM group. The present analysis demonstrated that the injection of MSC-CM into rats with DFUs enhanced the wound-healing process by accelerating wound closure, promoting cell proliferation and angiogenesis, enhancing cell autophagy, and reducing cell pyroptosis in ulcers. Conclusions: Studies conducted indicate that MSC-CM administration could be a novel cell-free therapeutic approach to treat DFUs accelerating the wound healing process and avoiding the risk of living cells therapy. Objectives marrowderived CellConditioned Cell Conditioned MSCCM MSC CM (MSC-CM Methods TDM T highfat high fat (DFUs n group, , group) ie i e i.e. NC, NC (NC) DMC, DMC C (DM-C) . (MSCs) PBS closure reepithelialization re epithelialization skin HE H E staining, staining staining) Ki67 Ki (Ki67 IHC, IHC IHC) CD31, CD (CD31 IFC) LC3B, LCB LC B (LC3B autolysosome, autolysosome autoly sosome, sosome autoly-sosome EM IL1β, IL1β ILβ IL 1β, 1β β (IL-1β NLRP3 NLRP Caspase1, Caspase1 Caspase 1, 1 Caspase-1 GSDMDN, GSDMDN N, N evaluated Results rate ELISA CD3 woundhealing Conclusions cellfree free therapy i.e (NC (DM-C (MSCs Ki6 (Ki6 (CD3 Caspase- (Ki (CD