Using fluorescence resonance energy transfer spectroscopy we demonstrate that thymosin β4 (tβ4) binding induces spatial rearrangements within the small domain (subdomains 1 and 2) of actin monomers in solution. Tβ4 binding increases the distance between probes attached to Gln-41 and Cys-374 of actin by 2 Å and decreases the distance between the purine base of bound ATP (εATP) and Lys-61 by 1.9 Å, whereas the distance between Cys-374 and Lys-61 is minimally affected. Distance determinations are consistent with tβ4 binding being coupled to a rotation of subdomain 2. By differential scanning calorimetry, tβ4 binding increases the cooperativity of ATP-actin monomer denaturation, consistent with conformational rearrangements in the tβ4-actin complex. Changes in fluorescence resonance energy transfer are accompanied by marked reduction in solvent accessibility of the probe at Gln-41, suggesting it forms part of the binding interface. Tβ4 and cofilin compete for actin binding. Tβ4 concentrations that dissociate cofilin from actin do not dissociate the cofilin-DNase I-actin ternary complex, consistent with the DNase binding loop contributing to high-affinity tβ4-binding. Our results favor a model where thymosin binding changes the average orientation of actin subdomain 2. The tβ4-induced conformational change presumably accounts for the reduced rate of amide hydrogen exchange from actin monomers and may contribute to nucleotide-dependent, high affinity binding.