[Abstract] Objective: To analyze tumor-associated neutrophils (TANs) infiltration in EBV-positive nasopharyngeal carcinoma (NPC) tumor microenvironment (TME) and its correlation with the prognosis and clinicopathological features of patients with NPC, and to preliminarily investigate the role of TANs in activating CD8+ T cells in EBV-positive NPC. Methods: Tumor tissue specimens from 118 patients with EBV-positive NPC without metastases admitted to Sun Yat-sen University Cancer Center from 2008 to 2012 were collected. Immunohistochemistry was performed to analyze CD8+ T cells infiltration and TANs infiltration in NPC and their correlation with EBV infection, and to evaluate the infiltration of TANs in 118 human EBV-positive NPC tissue samples and analyze its correlation with the prognosis and clinicopathological features of NPC patients; flow cytometry was used to detect the expression of N2 marker (CD182 and CD206) of neutrophils stimulated by HK1-EBV-conditioned medium and the expression of CD69 and PD-1 of CD8+ T cells co-cultured with TANs. Results: There were more CD8+ T cells and neutrophils infiltrating in EBV-positive NPC where they were negatively correlated（P=0.0052）; densely infiltrated TANs in EBV-positive NPC tissues were positively correlated with poor prognosis of patients (P=0.025 in OS; P=0.027 in PFS); the infiltration of TANs was proved to be an independent risk factor for overall survival (P=0.035) in patients with NPC; compared with Control group, freshly isolated neutrophils cultured in HK1-EBV-conditioned medium showed an increased expression of N2 markers (P<0.001 in CD182; P<0.01 in CD206) which could suppress the expression of CD69 and promote the expression of PD-1 of CD8+ T cells. Conclusion: More TANs infiltrate in EBV-positive NPC where they polarize into N2 neutrophils, exerting an immunosuppressive effect by suppressing the activation of CD8+ T cells and having a positive correlation with the poor prognosis of NPC patients.