<p>Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is an acute respiratory disorder for which there are no approved drugs with proven efficacy in its treatment. Liang-Ge-San (LGS), a classic Chinese prescription, is generally employed for treating respiratory diseases, including ALI. To elaborate on the therapeutic benefits and potential mechanism of LGS against ALI, the function of exosomal miR-21 in intercellular communication after LGS treatment was assessed by constructing the co-culture system between RAW 264.7 and the mouse type II alveolar epithelial (MLE-12) cells. LPS was employed through intratracheal instillation for constructing an ALI mice model to study the effect of LGS-stimulated exosomal miR-21. LGS suppressed the levels of pro-inflammatory cytokines and enhanced the expression of miR-21 in RAW 264.7 cells, which could be counteracted by the inhibition of miR-21. Moreover, LGS treatment enhanced the secretion of exosomes (referred to as LGS-exosomes) in RAW 264.7 cells, which were enriched with miR-21. MLE-12 cells were capable of internalizing LGS-exosomes, which was inhibited by exosome secretion inhibitor GW4869. Dual luciferase reporter assay exhibited direct PTEN mRNA inhibition by miR-21. LGS-exosomes markedly increased the protein expression of PI3K and p-AKT (Ser473), and decreased the protein expression of PTEN, as well as the pro-inflammatory cytokines in MLE-12 cells. In LPS-induced ALI mice, LGS-exosomes alleviated lung injury and edema, while simultaneously boosting the levels of miR-21 and regulating the proteins involved in the PTEN/PI3K/AKT pathway. LGS protects against LPS-induced ALI by upregulating macrophage-secreted exosomal miR-21 and subsequently regulating the PTEN/PI3K/AKT signaling pathway. Our study offers valuable insights for clinical application of LGS to treat ALI.</p> Graphical Abstract <p></p>