Many signaling pathways regulate the function of the cellular cytoskeleton. Yet we know very little about the proteins involved in the cross-talk between the signaling and the cytoskeletal systems. Here we show that myosin II-B, an important cytoskeletal protein, resides in a complex with p21-activated kinase 1 (PAK1) and atypical protein kinase C (PKC) zeta (aPKCζ) and that the interaction between these proteins is EGF-dependent. We further show that PAK1 is involved in aPKCζ phosphorylation and that aPKCζ phosphorylates myosin II-B directly on a specific serine residue in an EGF-dependent manner. This latter phosphorylation is specific to isoform B of myosin II, and it leads to slower filament assembly of myosin II-B. Furthermore, a decrease in aPKCζ expression in the cells alters myosin II-B cellular organization. Our finding of a new signaling pathway involving PAK1, aPKCζ, and myosin II-B, which is implicated in myosin II-B filament assembly and cellular organization, provides an important link between the signaling system and cytoskeletal dynamics.