Nivolumab, an anti-programmed death-1 (PD-1) monoclonal antibody, showed promising activity in relapsed or refractory (R/R) follicular lymphoma (FL) in a phase 1 study. We conducted a phase 2 trial to evaluate further its efficacy and safety in patients with R/R FL and to explore biomarkers of response. Patients with R/R FL and at least two prior lines of therapy, each containing a CD20 antibody or an alkylating agent, were treated with nivolumab 3 mg/kg every 2 weeks. The primary endpoint was objective response rate (ORR) assessed by independent radiologic review committee. Biomarker analyses included gene expression profiling and multiplex immunofluorescence studies of pretreatment tumor samples. A total of 92 patients were treated. After a minimum follow-up of 12 months, ORR was 4% (4/92). Median PFS was 2.2 months (95% confidence interval [CI], 1.9-3.6). Median DOR was 11 months (95% CI, 8-14). Exploratory analyses suggested that responders had significantly higher proportion of CD3+ T cells in the tumor microenvironment than non-responders, but no significant differences in PD-1 or PD-L1 expression were observed. High expression of a set of tumor-associated macrophage genes was associated with reduced PFS (hazard ratio, 3.28; 95% CI, 1.76-6.11; P = .001). The safety profile was consistent with previous reports of nivolumab. In conclusion, nivolumab monotherapy was associated with very limited activity in patients with R/R FL. Better understanding of the immune biology of this disease may facilitate the development of effective checkpoint-based strategies. This trial was registered at www.clinicaltrials.gov as #NCT02038946.