Vulvar squamous cell carcinoma (vSCC), although rare, carries significant morbidity and a high rate of recurrence. Treatment options beyond surgical excision remain limited. Lymphocyte activation gene-3 (LAG-3) and its binding partner galectin-3 (GAL-3) are an immuno-inhibitory checkpoint pair that represent potential immunotherapy targets for the treatment of vSCC. This study examined the expression of LAG-3 and GAL-3 alongside programmed cell death ligand-1 expression in invasive SCC and vulvar intraepithelial neoplasia (VIN) by immunohistochemical analysis of formalin-fixed paraffin-embedded tissue. A total of 35 cases were selected for evaluation: 13 VIN3 [human papillomavirus (HPV)-associated VIN/usual-type VIN], 2 differentiated VIN (dVIN), 16 HPV-associated vSCC, and 4 dVIN-associated vSCC. LAG-3+ tumor-infiltrating lymphocytes were identified in 91% (32/35) of cases of vulvar squamous neoplasia. Tumor cells were positive for GAL-3 in 71% of the vulvar neoplasia cases. HPV-associated vSCC was more likely to demonstrate GAL-3 tumoral positivity when compared with dVIN-associated vSCC (24/29 vs. 1/6, P=0.004). We observed co-expression of all 3 immunomarkers in 40% (14/35) of cases evaluated. In light of these findings, use of immunomodulatory drugs that target the LAG-3/GAL-3 pathway may be potentially beneficial in vSCC and efficacy may be increased when combined with anti-programmed cell death ligand-1 therapy.