BACKGROUND: The immunosuppressive tumor microenvironment (TME) in lung cancer, driven in part by M2-polarized tumor-associated macrophages (TAMs), contributes to worse prognosis and supports tumor progression. Cinobufagin (CB), an active compound in cinobufotalin injections, has demonstrated potential antitumor effects by modulating macrophage activity. This study investigated the mechanism by which CB influences glucose metabolism and polarization in M2 TAMs by focusing on the regulation of HIF-1α. METHODS: Human THP-1 monocytes were differentiated into M2 macrophages by stimulation with interleukin-4 at 20 ng/mL and then treated with cinobufagin at 2 μM, either alone or together with the HIF-1α activator DMOG at 1 mM. HIF-1α hydroxylation and ubiquitination were evaluated by Western blot and co-immunoprecipitation. Glycolytic activity was determined by measuring uptake of the glucose analogue 2-NBDG, extracellular lactate levels and expression of GLUT1, PKM2, LDHA and MCT1. M2 polarization markers CD206, Arg-1 and IL-10 were quantified by qRT-PCR, and TGF-β and IL-10 secretion was measured by ELISA. PD-L1 expression was assessed by Western blot, immunofluorescence and chromatin immunoprecipitation. Finally, conditioned media from treated macrophages were applied to A549 cells to evaluate migration through wound-healing assays and invasion using Transwell inserts, and to HUVECs to quantify tube formation. RESULTS: Using DMOG, an HIF-1α activator, we stimulated glycolysis in M2 macrophages, promoting their immunosuppressive polarization and elevating PD-L1 expression, a checkpoint protein associated with immune evasion. CB treatment reversed this effect by increasing HIF-1α hydroxylation and ubiquitination, leading to decreased HIF-1α stability, glucose uptake, and lactate production in M2 macrophages. Additionally, CB pre-treatment of M2 macrophages reduced the secretion of the cytokines TGF-β and IL-10, thereby limiting lung cancer cell migration, invasion, and angiogenesis. CONCLUSION: These findings suggest that CB suppresses M2 macrophage-mediated tumor support by targeting HIF-1α and glycolysis, thereby reprogramming the TME toward an anti-tumor state. This highlights CB's potential of CB in the treatment of lung cancer by countering immunosuppressive macrophage activity.