Actin dynamics in nonmuscle cells is controlled by the availability of actin nucleating sites and actin monomers. Thymosin β-4 (Tβ-4) has been implicated in modulating the availability of actin monomers in a large variety of cells. It together with actin nucleating, severing, and uncapping proteins, harnesses the intrinsic dynamic properties of actin to regulate the actin polymerization response in cells. Overexpression or addition of exogenous Tβ-4 or its homolog, Tβ-10, alters the actin cytoskeleton, and has multiple effects on cellular functions related to motility. Some of these effects are consistent with β-thymosins functioning exclusively as monomer-binding proteins, while others are not. Therefore, the complex pleiotropic effects of β-thymosin in cells may be due to direct and indirect effects on the actin cytoskeleton, as well as modulation of signaling pathways that will impact the cytoskeleton and a variety of cell functions.