At present, although the early treatment of sepsis is advocated, the treatment effect of sepsis is unsatisfactory, and the mortality rate remains high. Shenfu injection (SFI) has been used to treat sepsis with good clinical efficacy. Based on network pharmacology, this study adopted a new research strategy to identify the potential therapeutic targets and key active ingredients of SFI for sepsis from the perspective of the pathophysiology of sepsis. This analysis identified 28 active ingredients of SFI based on UHPLC-QQQ MS, including 18 ginsenosides and 10 aconite alkaloids. 59 targets were associated with the glycocalyx and sepsis pathways. Based on the number of targets related to the pathophysiological process of sepsis, we identified songorine, ginsenoside Rf, ginsenoside Re, and karacoline as the key active ingredients of SFI for the treatment of sepsis. According to the cluster analysis of MCODE and the validation on the GEO dataset, LGALS3, BCHE, AKT1, and IL2 were identified as the core targets. This study further explored the therapeutic mechanism and the key active ingredients of SFI in sepsis and provided candidate compounds for drug development. © 2022 Huajing Yuan et al.