Current limited evidence suggests that the use of pembrolizumab combined with chemotherapy may be effective for treatment-naïve patients with metastatic non-small cell lung cancer (NSCLC) and negative programmed cell death ligand 1 (PD-L1) expression, but real-world data are relatively scarce. This retrospective cohort study analyzed the efficacy, adverse events, and prognostic factors in these patients treated with chemotherapy with or without pembrolizumab.This retrospective study analyzed the data of patients with unresectable, locally advanced or metastatic NSCLC without sensitive epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) or proto-oncogene receptor tyrosine kinase (ROS1) alterations who had negative PD-L1 expression and received first-line pembrolizumab plus chemotherapy (the Pembro group) or platinum-based dual chemotherapy with or without bevacizumab (the Chemo group). The efficacy outcomes and safety profiles of the two groups were compared.The study included 246 patients (Pembro group, n=114; Chemo group, n=132). The median follow-up period was 28.3 months. The Pembro group significantly prolonged progression-free survival (PFS) compared with the Chemo group [9.5 months, 95% confidence interval (CI): 7.5-11.5 vs. 7.2 months, 95% CI: 5.7-8.7; hazard ratio (HR) =0.64, 95% CI: 0.46-0.87; P=0.004]. Squamous cell lung cancer (SCC) patients demonstrated substantial PFS benefit (13.8 months, 95% CI: 3.2-24.1 vs. 4.8 months, 95% CI: 3.4-6.2; P<0.001), while non-SCC patients showed comparable PFS (9.3 months, 95% CI: 7.6-11.0 vs. 8.0 months, 95% CI: 6.0-10.0; P=0.56). Overall survival (OS) favored the Pembro group (21.2 months, 95% CI: 16.0-26.4 vs. 20.1 months, 95% CI: 15.5-24.7; HR =0.71, 95% CI: 0.50-1.00; P=0.052). The SCC patients in the Pembro group demonstrated a significant survival benefit with a median OS that was not reached, compared to 14.2 months (95% CI: 6.3-22.1) in the chemo group (HR =0.42, 95% CI: 0.22-0.78; P=0.007). Grade ≥3 non-immune-related adverse events (non-irAEs) occurred more often in the Pembro group (46.8%) than the Chemo group (33.1%, P=0.03). Moreover, 45 (39.5%) patients experienced 63 irAEs, and no grade 5 or new irAEs were observed.Pembrolizumab combined with chemotherapy may prolong survival in patients with PD-L1-negative advanced NSCLC, particularly those with squamous histology.Copyright © 2025 AME Publishing Company. All rights reserved.