BACKGROUND: Excessive endothelial pro-inflammatory response is an early hallmark of sepsis-induced acute lung injury (ALI). Xuebijing (XBJ), a traditional Chinese medicine, is widely used in clinical practice to treat sepsis. PURPOSE: This study aims to investigate the molecular mechanisms underlying the beneficial effects of XBJ. METHODS: Plasma samples from septic patients treated with or without XBJ were collected and analyzed. The mouse model of sepsis was established by intraperitoneal injection of LPS (10 mg/kg). XBJ (10 ml/kg) was administrated intraperitoneally twice before LPS challenge and one time after LPS challenge. The severity of lung injury and the levels of inflammation and coagulation were evaluated. In vitro, HUVEC were used to explore the mechanisms of XBJ and its compounds in regulating the ACLY/MYB/RIG-I axis. RESULTS: XBJ significantly reduced the plasma levels of endothelial cell (EC) damage-related markers in septic patients. The in vivo and in vitro data demonstrated that XBJ alleviated LPS-induced lung injury and reduced the levels of inflammation and coagulation activation in ECs. XBJ inhibited the phosphorylation-dependent activation of ATP citrate lyase (ACLY), thereby suppressing the acetylation-dependent nuclear translocation of the transcription factor MYB. The expression of retinoic acid inducible gene I (RIG-I) was downregulated, leading to the inhibition of NF-κB signaling and EC pro-inflammatory and coagulation activation, which further alleviated sepsis-associated ALI. Moreover, XBJ compounds Quercetin, Ferulic Acid, Kaempferol and Paeoniflorin all showed inhibitory effects on the activation of the downstream MYB/RIG-I signaling by binding to ACLY protein. CONCLUSION: Our study revealed a novel regulatory mechanism of XBJ in sepsis-induced EC dysfunction and ALI. The compounds in XBJ inhibited the activity of ACLY, thereby inhibiting the expression of RIG-I by reducing the acetylation of transcription factor MYB, leading to the alleviation of EC activation and lung injury induced by sepsis. Our findings provide a theoretical basis for the clinical application of XBJ and shedding light on novel therapeutic targets for treating sepsis.