Acute lung injury (ALI) is a frequent and challenging aspect of sepsis that currently lacks effective treatments. Procyanidin B2 (PB2) has anti-inflammatory and antioxidant properties. The aim of this study was to determine the effectiveness and mechanism of action of PB2 in treating sepsis-induced ALI using animal experiments. A sepsis-induced ALI mouse model was used by administering lipopolysaccharide (LPS) and then evaluating the levels of inflammatory cytokines and lung injury through measurements of cytokine levels using enzyme-linked immunosorbent assay (ELISA), Western blot and real-time PCR, as well as by the examination of relevant signaling pathways. The animal experiments showed that PB2 protected the lungs from injury caused by LPS and reduced the levels of various inflammatory cytokines in both the serum and lung tissue. Western blot analysis showed that PB2 reduced the expression of TLR4/NF-κB and increased the expression of PI3K/Akt, and also inhibited the Hippo and Rho signaling pathways. The results of the study showed that PB2 helps to treat sepsis-induced ALI by controlling cytokine storms and reducing inflammation by altering the expressions of the TLR4/NF-κB, PI3K/Akt, Hippo and Rho signaling pathways. This research provides a foundation for the further investigation of PB2’s mechanism and its potential use in treating sepsis.