Purpose Delayed skin healing in diabetic wounds is a major clinical problem. The tRNA-derived small RNAs (tsRNAs) were reported to be associated with diabetes. However, the role of tsRNAs in diabetic wound healing is unclear. Our study was designed to explore the tsRNA expression profile and mine key potential tsRNAs and their mechanism in diabetic wounds. Methods Skin tissues of patients with diabetic foot ulcers and healthy controls were subjected to small RNA sequencing. The role of candidate tsRNA was explored by loss- and gain-of-function experiments in HUVECs. Results A total of 55 differentially expressed tsRNAs were identified, including 12 upregulated and 43 downregulated in the diabetes group compared with the control group. These tsRNAs were mainly concentrated in intercellular interactions and neural function regulation in GO terms and enriched in MAPK, insulin, FoxO, calcium, Ras, ErbB, Wnt, T cell receptor, and cGMP-PKG signaling pathways. tRF-Gly-CCC-039 expression was upregulated in vivo and in vitro in the diabetic model. High glucose disturbed endothelial function in HUVECs, and tRF-Gly-CCC-039 mimics further harmed HUVECs function, characterized by the suppression of proliferation, migration, tube formation, and the expression of Coll1a1, Coll4a2, and MMP9. Conversely, the tRF-Gly-CCC-039 inhibitor could attenuate high-glucose-induced endothelial injury to HUVECs. Conclusion We investigated the tsRNAs expression profile in diabetic foot ulcers and defined the impairment role of tRF-Gly-CCC-039 in endothelial function in HUVECs. This study may provide novel insights into accelerating diabetic skin wound healing.