Receptor for advanced glycation end products (RAGE) is implicated in tumor biology. Released high mobility group box protein 1 (HMGB1) ligand binding to RAGE receptor in tumor cells promotes tumor progression. The mechanisms of HMGB1-RAGE signaling in M2 macrophages involved in lymphangiogenesis in laryngeal carcinoma remain poorly understood. Here, we assessed the effect of HMGB1-RAGE signaling on M2 macrophages in lymphangiogenesis.HMGB1, CD163, and D2-40 in laryngeal squamous cell carcinoma (LSCC, n = 123), laryngeal precursor lesions (LPLs, n = 102), and vocal polyp (VP, n = 55) were analyzed by immunohistochemistry. THP-1 cell-expressed RAGE gene was knocked down and then polarized to M0 macrophages and M2 macrophages. IL-23, TNF-α, TGF-β, and IL-10 were measured by ELISA; IL-1β, IL-12, IL-10, and CCL-13 were evaluated by RT-qPCR, and CD206, CD163, and RAGE were evaluated by western blot to evaluate whether classical M2 macrophages were obtained. Conditioned media from RAGE+/- M0 macrophages and RAGE+/- M2 macrophages incubated in the presence or absence of HMGB1, anti-Toll-like receptor (TLR)2, anti-TLR4 antibodies, and anti-VEGF-C antibodies were collected separately for human dermal lymphatic endothelial cells (HDLEC) for proliferation, migration, lymphangiogenesis assay, and VEGF-C concentration analysis.HMGB1 and M2 macrophage densities were increased in LSCC (P < 0.01). HMGB1 and M2 macrophage densities were significantly correlated with lymphatic vessel density (LVD) in LSCC (P < 0.01). The HMGB1 overexpression and higher M2 macrophage density were involved in lymph node metastasis (P < 0.01) and poor prognosis (P < 0.05). In vitro, conditioned medium from HMGB1-stimulated RAGE+ M2 macrophages activated lymphangiogenesis by upregulating the VEGF compared to controls (P < 0.05). On the contrary, RAGE knockdown obviously decreased the corresponding effects of HMGB1-preconditioned M2 macrophages upon HDLEC (P < 0.05). HMGB1-TLR pathway does not significantly increase HDLEC proliferation, migration, and lymphangiogenesis on M2 macrophages.HMGB1 promotes lymphangiogenesis by activation of RAGE on M2 macrophages. Targeting RAGE may provide an effective therapeutic strategy against M2 macrophages in LSCC patients with lymph node metastasis.Copyright © 2022 Caili Su et al.