Damage to the integrity of heparin sulfate (HS) in the endothelial glycocalyx is an important factor of glomerular filtration barrier dysfunction, which is the basic pathological feature of acute kidney injury (AKI). AKI is a common clinical critical illness with few drugs options offering effective treatment. Phillyrin (Phil), the main pharmacological component of Forsythia suspensa, possesses a wide range of pharmacological activities. However, the effects of Phil on lipopolysaccharide (LPS)-induced AKI have yet to be reported. The aim of the present study is to analyze the effects of Phil on HS damage and inflammatory signaling pathways in LPS-induced AKI. Results revealed that Phil reduces pathological changes and improves renal function in LPS-induced AKI. Further analysis indicated that Phil effectively protects against glycocalyx HS degradation in LPS-stimulated EA.hy926 cells in vitro and LPS-induced AKI mice in vivo. The protective effect of Phil on HS damage may be associated with the isolate's ability to suppress the production of reactive oxygen species, and decrease expression levels of cathepsin L and heparanase in vitro and in vivo. In addition, ELISA and Western blot results revealed that Phil inhibits the activation of the NF-κB and MAPK signaling pathways and decreases the levels of inflammatory cytokines (IL-1β, IL-6, and TNF-α) in LPS-induced ARDS mice. In general, protection against endothelial glycocalyx HS damage and inhibition of inflammatory responses by Phil may be used as treatment targets for LPS-induced AKI.