Background: As a novel treatment, programmed cell death protein 1 (PD-1) inhibitor appears to be less effective in tumors of lung adenocarcinoma patients with epidermal growth factor receptor (EGFR) mutation. Beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) has reported to be associated with programmed death ligand 1 (PD-L1)/PD-1 interaction. However, the relationship between B3GNT3 and PD-L1 and its prognostic significance in EGFR-mutant status are still unknown. Methods: B3GNT3 was identified through transcriptome sequencing and The Cancer Genome Atlas Lung Adenocarcinoma (TCGA-LUAD) database. Flow cytometry and real-time polymerase chain reaction were performed to investigate the association between B3GNT3, PD-L1, and EGFR. Then, B3GNT3 and PD-L1 expression were evaluated by immunohistochemical analysis in 145 surgically resected primary lung adenocarcinomas. The relationships between survival and B3GNT3, PD-L1, and EGFR status were assessed, and the potential prognostic factors in patients with B3GNT3 expression were identified. Results: We found that EGFR activation induced PD-L1 expression, and EGFR tyrosine kinase inhibitor (TKI) could reduce PD-L1 protein in EGFR-TKI-sensitive HCC827 and PC9 cell lines. Subsequent analysis showed that EGFR inhibitor could also lead to both decreased PD-L1 and B3GNT3 mRNA expression. A total of 145 lung adenocarcinoma patients were included. PD-L1 >1% and B3GNT3-positive expression in patients might contribute to worse prognosis in both overall survival (OS) [hazard ratio (HR), 2.63; 95% confidence interval (CI), 0.98-7.06; P=0.048] and disease-free survival (DFS) (HR, 3.04; 95% CI, 1.13-8.14; P=0.019), especially in the PD-L1 >= 50% group. However, when patients were negative for B3GNT3, PD-L1, and EGFR (or "triple negative"), there were significant decreases in OS (HR, 5.44; 95% CI, 0.99-29.83; P=0.029) and DFS (HR, 7.24; 95% CI, 1.32-39.73; P=0.008). Positive B3GNT3 expression was a significant risk factor associated with lower DFS (HR, 3.30; P=0.043). Conclusions: Our results indicate that the B3GNT3 expression is tightly correlated with PD-L1 expression and EGFR mutation status. B3GNT3 is associated with poor prognosis in lung adenocarcinoma patients. Collectively, these findings may offer new insight into enhancing immune therapy efficacy for lung adenocarcinoma patients.