Objective ·To investigate the role and mechanism of sirtuin 5 (SIRT5) in pulmonary microvascular endothelial cell injury in sepsis. Methods ·Wild-type (WT) and Sirt5 gene knockout C57BL/6 male mice underwent cecal ligation and puncture (CLP) surgery. Following euthanasia, lung tissues were collected. Pulmonary inflammation was assessed using hematoxylin and eosin (H-E) staining; vascular leakage was evaluated by Evans blue (EB) staining; coagulation function in mice was analyzed via immunofluorescence staining of lung tissues. Immunohistochemical staining was employed to detect vascular cell adhesion molecule-1(VCAM-1) protein expression, thereby assessing endothelial inflammation in CLP-treated mice. By using gene editing technology, SIRT5 was knocked down or overexpressed in human umbilical vein endothelial cells (HUVECs), and the cells were subsequently stimulated with lipopolysaccharide (LPS) to induce endothelial inflammation. Protein expression levels of VCAM-1, tissue factor (TF), and other endothelial injury markers were detected by Western blotting, and inflammatory cytokines such as interleukin-6 (IL-6) and IL-1β, were detected by quantitative real-time PCR (qPCR). In addition, transcriptomic sequencing was performed on HUVECs overexpressing SIRT5, and key genes including F2R-like thrombin or trypsin receptor 3 (F2RL3), serpin family A member 3 (SERPINA3), and transforming growth factor β2/β3 (TGF-β2/3) were validated by qPCR. Results ·Sirt5 knockout significantly aggravated lung injury in CLP mice, reducing their survival rates (P<0.001). H-E staining showed increased inflammatory infiltration in the lung tissue of the mice, while EB staining indicated increased vascular leakage (P<0.001). Immunofluorescence revealed elevated fibrinogen deposition. In HUVECs with SIRT5 knockdown, the protein levels of VCAM-1 and TF, as well as the mRNA levels of inflammatory factors including IL-6, IL-1β, VCAM-1, and E-selectin, were significantly upregulated (all P<0.001), whereas overexpression of SIRT5 reversed these effects. Transcriptome sequencing analysis indicated that SIRT5 regulated endothelial inflammation and coagulation responses by inhibiting the F2RL3/SERPINA3/TGF‑β pathway. Conclusion ·SIRT5 negatively regulates the F2RL3/SERPINA3/TGF‑β signaling axis, thereby alleviating endothelial inflammation and promoting coagulation responses, suggesting its potential protective role in sepsis-induced lung injury.