BACKGROUND Immunotherapy has shown promising efficacy in patients with nasopharyngeal carcinoma (NPC). Lymphocyte activating 3 gene (LAG-3) represents a significant immune target, however, its relationship with NPC remains unclear. This study aimed to evaluate LAG-3 expression in NPC and its association with CD3+ tumor-infiltrating lymphocytes (TILs), Granzyme B (GZMB), programmed death ligand 1 (PD-L1), and programmed death 1 (PD-1) expression. METHODS A total of 182 patients with NPC from Sun Yat-sen University Cancer Center, China, were included in this retrospective study. LAG-3 expression in 15 NPC cell lines and LAG-3, CD3+ TILs, GZMB, PD-L1 and PD-1 in clinical samples were estimated using immunohistochemistry. The Chi-square test was used to estimate the association between LAG-3, other biomarkers, and clinical characteristics. Survival analysis was performed using the Kaplan-Meier method and the Cox regression model. RESULTS LAG-3 was negatively expressed in all of the 15 NPC cell lines, whereas, 147 patients with NPC (80.8%) exhibited high LAG-3 expression on TILs from tumor tissues. Male patients and those who were EBV-positive presented higher LAG-3 expression. Correlation analyses showed that LAG-3 expression was related to PD-1 expression on TILs, as well as, PD-L1 expression on tumor cells (TCs) and TILs. Both the univariate and multivariate Cox models indicated that pathological type III (P = 0.036), higher LAG-3 on TILs (P < 0.001), higher PD-L1 on TCs (P = 0.027), and higher PD-1 on TILs (P < 0.001) were associated with poorer disease-free survival (DFS). However, lower PD-L1 expression on TILs was related to superior DFS only in the univariate Cox analyses (P = 0.002). CONCLUSIONS Higher LAG-3 and PD-1 on TILs, and higher PD-L1 expression on TCs, and pathological type III were identified as independent risk factors for poorer DFS in NPC patients. Our data demonstrate that LAG-3 is a promising inhibitory receptor that may play an important role in anti-NPC therapy.