Acute lung injury (ALI) is a severe organ dysfunction caused by sepsis. WIN55212-2 (WIN) is a cannabinoid receptor agonist. Activation of cannabinoid type 2 receptor can alleviate septic lung injury. Therefore, the effects of WIN on sepsis-related ALI were evaluated.MiR-29b-3p, FOXO3 and PFKFB3 levels, as well as M1 and M2 macrophage markers were assessed by RT-qPCR in MH-S cells after lipopolysaccharide (LPS) and WIN treatment. ChIP and dual luciferase reporter assays determined molecules interactions. Glycolysis-related proteins were evaluated by Western blotting assay. Lactic acid and ATP were also tested. Furthermore, the effect of WIN was tested in sepsis mice model. HE staining evaluated the histopathological changes in mouse lung tissues. The number of inflammatory cells and macrophages, protein concentration and lactic acid content were detected in mouse bronchoalveolar lavage fluid.We found that WIN suppressed M1 polarization and glycolysis in alveolar macrophages induced by LPS. Moreover, WIN inhibited FOXO3 by up-regulating miR-29b-3p. Furthermore, we verified that FOXO3 induced macrophage M1 polarization and glycolysis through activating PFKFB3. In vivo, WIN alleviated ALI in mice with sepsis.Our results reveal that WIN inhibits macrophage glycolysis through the miR-29b-3p/ FOXO3/PFKFB3 axis, suggesting new therapeutic targets to alleviate sepsis-related ALI.Copyright © 2022 Elsevier Ltd. All rights reserved.