Background and Aims Peripheral arterial disease (PAD) is a condition characterized by insufficient blood flow due to various reasons, and some patients are not suitable for surgery. In recent years, stem cell transplantation has been used in the treatment of PAD, showing promising results. However, the metabolic mechanisms involved in its therapeutic effects remain unclear. This study was conducted to investigate the metabolic pathways and molecules involved in the repair of lower limb ischemic tissue by human umbilical cord mesenchymal stem cells (HUCMSCs) using liquid chromatography-mass spectrometry (LC-MS) metabolomics, with a specific focus on changes in the acid sphingomyelinase (ASM)-ceramide (Cer) metabolic pathway.Methods HUCMSCs were isolated from human umbilical cord tissue, expanded in culture, and characterized using flow cytometry for surface molecular markers. A mouse model of lower limb ischemia was created by ligating and excising the left femoral artery and vein in 8-week-old male C57BL/6J mice. Then, the mice were randomly divided into two groups, with one group receiving local injection of HUCMSCs suspension (HUCMSCs group) and the other receiving PBS (control group). Muscle tissues from the ischemic limb were collected at 3, 7, and 14 d after surgery. HE staining and Masson staining were performed to observe morphological changes. LC-MS analysis was conducted in combination with KEGG database analysis to compare the metabolomic differences between the two groups.Results HUCMSCs expressed high levels of CD105, CD90, and CD73, and low levels of HLA-DR, CD14, CD19, and CD34. Morphological observations revealed a significant improvement in muscle atrophy and fibrosis in the HUCMSCs group compared to the control group at 7 and 14 d after surgery. Non-targeted metabolomic analysis identified 687 metabolites in the gastrocnemius muscle samples, with lipids representing the largest proportion (34.088%). Differential metabolites included 37 in negative ion mode (25 upregulated, 12 downregulated) and 17 in positive ion mode (11 upregulated, 6 downregulated). Cer was significantly downregulated (FC=0.43), and the phosphatidylcholine product of the ASM/Cer pathway also decreased (FC=0.68) compared to the control group. KEGG pathway analysis of combined positive and negative ion mode data from gastrocnemius muscle samples at 3 and 7 d after surgery revealed involvement in pathways such as γ-aminobutyric acid-ergic synapse, arginine/proline metabolism, mineral absorption, oxidative phosphorylation, protein metabolism, glycerophospholipid metabolism, and regulation of the actin cytoskeleton.Conclusion Changes in lipid metabolism play a crucial role in the repair of lower limb ischemic injury promoted by HUCMSCs, with some mechanisms potentially associated with the inhibition of the ASM/Cer pathway.