Serine/threonine p21-activated kinase is an effector of Rac with a key role in the regulation of cytoskeletal organization. Non-muscle myosin II is a molecular motor, which is an important component of the cytoskeleton. Non-muscle myosin II-B plays a major role in cell motility and chemotaxis. We investigated the role of Rac and p21-activated kinase 1 (PAK1) in the regulation of myosin II-B in prostate cancer cells in response to epidermal growth factor (EGF) stimulation. We found that both Rac and PAK1 affect EGF-dependent non-muscle heavy chain II-B localization and cell morphology. We further found that a dominant negative mutant of PAK1 significantly inhibits EGF-dependent myosin II-B heavy chains phosphorylation and filament disassembly. Furthermore, cells expressing the dominant negative mutant exhibited an increase in EGF-dependent myosin light chain phosphorylation and diminished chemotaxis towards EGF. To our knowledge this is the first report exploring the role of PAK1 in the regulation of both non-muscle myosin II-B heavy chains and light chains. Furthermore, the data presented here suggest that PAK1 plays a crucial role in the regulation of cell morphology and chemotaxis by regulating the phosphorylation and cellular localization of myosin II-B.