Abstract Purpose: Although programmed death 1/programmed death ligand 1 (PD-1/PD-L1) inhibitors are promising agents for recurrent or metastatic nasopharyngeal carcinoma (NPC), PD-1/PD-L1 inhibitor monotherapy has shown modest efficacy. This study evaluated the efficacy and safety of nivolumab plus gemcitabine in patients with NPC who failed prior platinum-based chemotherapy. Patients and Methods: This is a phase II, multicenter, open-label, single-arm study. Patients with recurrent or metastatic NPC received nivolumab 3 mg/kg and gemcitabine 1,250 mg/m2 every 2 weeks until disease progression or intolerable toxicity. The primary endpoint was progression-free survival (PFS). The secondary endpoints included objective response rate (ORR), overall survival (OS), and safety. To identify potential biomarkers, whole-exome sequencing, whole-transcriptome sequencing, and immune phenotype analysis based on Lunit SCOPE IO, an artificial intelligence–powered spatial tumor-infiltrating lymphocyte analyzer, were performed. Results: Thirty-six patients were enrolled between June 2018 and June 2019. The ORR was 36.1% and disease control rate was 97.2%. With median follow-up of 22.0 months, median PFS was 13.8 months [95% confidence interval (CI), 8.6–16.8 months]. Median OS was not reached, and OS rate at 6 months was 97.0% (95% CI, 80.4%–99.6%). The grade ≥3 treatment-related adverse events were hypertension (2.8%) and anemia (2.8%). In multivariate analysis of mutation of chromatin modifier gene, tumor mutational burden (≥ 2.1 mut/Mb), and somatic copy-number alteration (SCNA) level, the group with high SCNA (> 3 points; HR, 7.0; 95% CI, 1.3–37.9; P = 0.02) had independently associated with poor PFS. Immune phenotype analysis showed that tumors with high proportion of immune-excluded immune phenotype was significantly correlated with poor PFS (HR, 4.4; 95% CI, 1.2–16.2; P = 0.018). Conclusions: Nivolumab plus gemcitabine showed promising efficacy with favorable toxicity profiles in patients with advanced NPC in whom platinum-based combination chemotherapy failed.