CD276 has been identified as a novel immune checkpoint, and its overexpression is associated with immune evasion and poor prognosis in various tumors, including head and neck squamous cell carcinoma (HNSCC). Nimotuzumab, a humanized anti-epidermal growth factor receptor (EGFR) monoclonal antibody, has been approved for various solid tumors. However, it remains unclear whether its anticancer efficacy involves a reduction in CD276 expression. The purpose of this study was to investigate the regulatory effects and potential mechanisms of nimotuzumab on CD276 expression both in vitro and in vivo. In a coculture system, nimotuzumab showed inhibitory effects on TGF-β-induced upregulation of CD276 at both the transcriptional and protein levels in HNSCC cell lines. Mechanistic studies revealed that nimotuzumab primarily suppressed TGF-β-induced CD276 upregulation by blocking EGFR/MEK/ERK, which was further validated by MEK and ERK inhibitors. In xenograft and mice HNSCC models, nimotuzumab exerted antitumor effects accompanied by significantly reduced CD276 expression during tumor progression. Analysis of tumor-infiltrating lymphocytes (TILs) profiles indicated that nimotuzumab orchestrated the tumor immune microenvironment (TIME) by notably increasing the frequency of T lymphocytes, including cytotoxic T lymphocytes and helper T lymphocytes, as well as macrophage cells. However, no significant changes were observed in the populations of NK cells, DC cells, and neutrophils. These findings offer new insights into the anticancer mechanisms of nimotuzumab and its underlying synergy in combined treatments with immunotherapy for HNSCC.Copyright © 2025 Elsevier B.V. All rights reserved.