Objective Treatment options for advanced vulvar cancer are limited. We evaluated pembrolizumab monotherapy in patients with advanced vulvar squamous cell carcinoma (SCC) enrolled in the phase 2 multicohort, open-label KEYNOTE-158 study (NCT02628067). Methods Eligible patients had histologically or cytologically documented advanced vulvar SCC with prior treatment failure, measurable disease per RECIST v1.1, ECOG performance status 0–1, and a tumor sample available for biomarker analysis. Pembrolizumab 200 mg was administered intravenously Q3W for up to 35 cycles (approximately 2 years). The primary endpoint was objective response rate (ORR) per RECIST v1.1 by independent central radiologic review in all patients and subgroups based on PD-L1 combined positive score (≥1 [PD-L1–positive] versus <1 [PD-L1–negative]). Results 101 patients were enrolled. Median time from first dose to data cutoff was 36.0 months. The ORR (95% CI) was 10.9% (5.6%–18.7%) among all patients, 9.5% (4.2%–17.9%) among the 84 patients with PD-L1–positive tumors, and 28.6% (3.7%–71.0%) among the 7 patients with PD-L1–negative tumors. Among patients with a response, median DOR was 20.4 (range, 2.1+ to 28.0) months. Median (95% CI) PFS and OS were 2.1 (2.0–2.1) and 6.2 (4.9–9.4) months, respectively. Treatment-related AEs occurred in 50.5% of patients (grade 3–5, 11.9%) and led to discontinuation of treatment in 5.0% of patients. Two deaths were considered treatment-related (hepatitis, n = 2). Conclusions Pembrolizumab monotherapy was associated with durable responses in a subset of patients with vulvar SCC. Responses occurred regardless of tumor PD-L1 status. No new safety signals emerged; overall, pembrolizumab was well tolerated.