radiotherapy stands as an important complementary treatment for head and neck squamous cell carcinoma (HNSCC), yet it does not invariably result in complete tumor regression. The infiltration of immunosuppressive macrophages is believed to mediate the radiotherapy resistance, which mechanism remains largely unexplored. This study aimed to elucidate the role of immunosuppressive macrophages during radiotherapy and the associated underlying mechanisms.Male C3H mice bearing syngeneic SCC-VII tumor were received irradiation (2 × 8Gy). The impact of irradiation on tumor-infiltrating macrophages were assessed. Bone marrow derived macrophages were evaluated in differentiation, proliferation, migration, and inflammatory cytokines after treatment of irradiated tumor culture medium (irCM) and irradiated tumor derived extracellular vesicles (irTEVs). A comprehensive metabolomics profiling of the irTEVs was conducted using liquid chromatography-mass spectrometry, while key metabolites were investigated the mechanism in macrophage in vitro and in vivo.Radiotherapy on SCC-VII syngeneic graft tumors increased polarization of both M1 and M2 macrophages in tumor microenvironment and drove infiltrated macrophages towards an immunosuppressive phenotype. Irradiation-induced polarization and immunosuppression of macrophages were dependent on irTEVs which delivered an increased amount of nicotinamide (NAM) to macrophages. NAM directly bound to the NF-κB transcriptional activity regulator USP7, through which NAM reduced translocation of NF-κB into the nucleus, thereby decreasing the release of cytokines IL6 and IL8. Increased enzyme activity of nicotinamide phosphoribosyl transferase (NAMPT) which is the rate-limiting enzyme of NAD+ metabolism, contributed to the irradiation-induced accumulation levels of NAM in irradiated HNSCC and irTEVs. Inhibition of NAMPT decreased NAM levels in irTEVs and increased radiotherapy sensitivity through alleviating immunosuppressive function of macrophages.Radiotherapy could induce NAD+ metabolic reprogramming of HNSCC cells, which regulate macrophage towards an immunosuppressive phenotype. Pharmacological targeting NAD+ metabolism might be a promising strategy for radiotherapy sensitization of HNSCC.Copyright © 2024 Elsevier Ltd. All rights reserved.