The programmed cell death protein-1 (PD-1)/programmed cell death ligand-1 (PD-L1) axis blockade has been implemented in advanced-stage tumor therapy for various entities, including head and neck squamous cell carcinoma (HNSCC). Despite a promising tumor response in a subgroup of HNSCC patients, the majority suffer from disease progression. PD-L1 is known to influence several intrinsic mechanisms in cancer cells, such as proliferation, apoptosis, migration and invasion. Here, we modulated PD-L1 expression in three HNSCC cell lines with differential intrinsic PD-L1 expression. In addition to an alteration in the epithelial-to-mesenchymal transition (EMT) marker expression, we observed PD-L1-dependent cell spreading, migration and invasion in a spheroid spreading assay on four different coatings (poly-L-lysine, collagen type I, fibronectin and Matrigel(R)) and a chemotactic transwell migration/invasion assay. Furthermore, the overexpression of PD-L1 led to increased gene expression and small interfering ribonucleic acid (siRNA) knockdown and decreased gene expression of Rho-GTPases and related proteins in a RT2 Profiler (TM) PCR Array. Rac1 and Rho-GTPase pulldown assays revealed a change in the activation state concordantly with PD-L1 expression. In summary, our results suggest a major role for PD-L1 in favoring cell motility, including cell spreading, migration and invasion. This is presumably caused by altered N-cadherin expression and changes in the activation states of small Rho-GTPases Rho and Rac1.