p21-activated kinases (PAKs) are a family of serine/threonine kinases that regulate cytoskeletal dynamics and cell motility. PAKs are subdivided into group I (PAKs 1–3) and group II (PAKs 4–6) on the basis of structural and functional characteristics. Based on prior gene expression data that predicted enhanced PAK signaling in the invasive fronts of aggressive papillary thyroid cancers (PTCs), we hypothesized that PAKs functionally regulate thyroid cancer cell motility and are activated in PTC invasive fronts. We examined PAK isoform expression in six human thyroid cancer cell lines (BCPAP, KTC1, TPC1, FTC133, C643, and SW1746) by quantitative reverse transcription-PCR and western blot. All cell lines expressed PAKs 1–4 and PAK6 mRNA and PAKs 1–4 protein; PAK6 protein was variably expressed. Samples from normal and malignant thyroid tissues also expressed PAKs 1–4 and PAK6 mRNA; transfection with the group I (PAKs 1–3) PAK-specific p21 inhibitory domain molecular inhibitor reduced transwell filter migration by ∼50% without altering viability in all cell lines ( P <0.05). BCPAP and FTC133 cells were transfected with PAK1, PAK2, or PAK3-specific small interfering RNA (siRNA); only PAK1 siRNA reduced migration significantly for both cell lines. Immunohistochemical analysis of seven invasive PTCs demonstrated an increase in PAK1 and pPAK immunoactivity in the invasive fronts versus the tumor center. In conclusion, PAK isoforms are expressed in human thyroid tissues and cell lines. PAK1 regulates thyroid cancer cell motility, and PAK1 and pPAK levels are increased in PTC invasive fronts. These data implicate PAKs as regulators of thyroid cancer invasion.