Tumor microenvironment (TME) cells orchestrate an immunosuppressive milieu that supports cancer cell proliferation. Tumor-associated neutrophils (TANs) have gained attention as inflammation biomarkers. However, the role of heterogeneous TAN populations in TME immune tolerance and their clinical potential remain unclear. Herein, we used public database to conduct single-cell transcriptomic analysis of 81 patients with non-small cell lung cancer (NSCLC) to elucidate TAN phenotypes linked to unfavorable clinical outcomes. We identified a pro-tumoral TAN cluster characterized by elevated HMGB1 expression, which could potentially engage with the TME through HMGB1-TIM-3 interaction. GATA2 was the transcription factor that drove HMGB1 expression in this pro-tumoral TAN subcluster. Further in vivo experiments confirmed the recruitment of HMGB1-positive TANs to the tumor lesion. Dual-luciferase reporter assays consolidated that the transcription factor GATA2 mediated HMGB1 expression by binding to its promoter region. Moreover, surgical NSCLC specimens verified the putative association between HMGB1-positive TAN and the pathological grades of primary tumors. Overall, this report revealed a pro-tumoral TAN cluster with HMGB1 overexpression that potentially dampen anti-tumoral immunity and contributed to immune evasion via the GATA2/HMGB1/TIM-3 axis. Moreover, this report suggests that this specific phenotype of TAN could serve as an indicator to clinical outcomes and immunotherapy effects for NSCLC.