The dynamic remolding of the actin cytoskeleton is a critical part of most cellular activities, and malfunction of cytoskeletal proteins results in various human diseases. The transition between two forms of actin, monomeric or G-actin and filamentous or F-actin, is tightly regulated in time and space by a large number of signaling, scaffolding and actin-binding proteins (ABPs). New ABPs are constantly being discovered in the post-genomic era. Most of these proteins are modular, integrating actin binding, protein-protein interaction, membrane-binding, and signaling domains. In response to extracellular signals, often mediated by Rho family GTPases, ABPs control different steps of actin cytoskeleton assembly, including filament nucleation, elongation, severing, capping, and depolymerization. This review summarizes structure-function relationships among ABPs in the regulation of actin cytoskeleton assembly.