Background Filamentous actin (F-actin) forms polymers that contribute to the abnormal biophysical properties of sputum. Thymosin β4 (Tβ4) is the major monomeric actin-sequestering peptide in cells and can depolymerize F-actin. Tβ4 could potentially decrease sputum viscoelasticity and adhesivity and improve sputum clearance. Methods Sputum was collected during pulmonary function testing from 17 subjects during a cystic fibrosis (CF) center visit. Sputum rheology, cough and ciliary transportability, and interfacial tension were measured before and after the addition of dornase alfa at 30 μg/mL; Tβ4 at 0.3, 3, 30, and 150 μg/mL; and Tβ4 with dornase alfa at 1.5 μg/mL each. Sputum was separately incubated with Tβ4 at 30 μg/mL for 0, 10, 20, or 60 min. Results There was a direct relationship between actin filament length and sputum cohesivity. There was a dose-dependent threshold decrease in cohesivity with Tβ4 and a time-dependent decrease in cohesivity over 60 min at 30 μg/mL. With the combination of dornase alfa and Tβ4 at 1.5 μg/mL each, there was a 70% decrease in G*s and a 65% decrease in G′ at 1 rad/s (p = 0.013). There was a 44% increase in cough transportability of sputum in vitro (p = 0.037) and a 71% increase in mucociliary transportability of sputum in vitro (p = 0.013) relative to control with the combination of dornase alfa and Tβ4, but no significant change with dornase alfa or Tβ4 alone at any concentration. Conclusions Actin polymer filament length is correlated with sputum cohesivity. Tβ4 depolymerizes CF sputum actin in both a dose-dependent and a time-dependent manner. An apparent synergy of Tβ4 on actin and dornase on DNA may be explained by the combined effect of actin depolymerization and DNA filament severing or by virtue of actin depolymerization increasing the effectiveness of dornase alfa. Filamentous actin (F-actin) forms polymers that contribute to the abnormal biophysical properties of sputum. Thymosin β4 (Tβ4) is the major monomeric actin-sequestering peptide in cells and can depolymerize F-actin. Tβ4 could potentially decrease sputum viscoelasticity and adhesivity and improve sputum clearance. Sputum was collected during pulmonary function testing from 17 subjects during a cystic fibrosis (CF) center visit. Sputum rheology, cough and ciliary transportability, and interfacial tension were measured before and after the addition of dornase alfa at 30 μg/mL; Tβ4 at 0.3, 3, 30, and 150 μg/mL; and Tβ4 with dornase alfa at 1.5 μg/mL each. Sputum was separately incubated with Tβ4 at 30 μg/mL for 0, 10, 20, or 60 min. There was a direct relationship between actin filament length and sputum cohesivity. There was a dose-dependent threshold decrease in cohesivity with Tβ4 and a time-dependent decrease in cohesivity over 60 min at 30 μg/mL. With the combination of dornase alfa and Tβ4 at 1.5 μg/mL each, there was a 70% decrease in G*s and a 65% decrease in G′ at 1 rad/s (p = 0.013). There was a 44% increase in cough transportability of sputum in vitro (p = 0.037) and a 71% increase in mucociliary transportability of sputum in vitro (p = 0.013) relative to control with the combination of dornase alfa and Tβ4, but no significant change with dornase alfa or Tβ4 alone at any concentration. Actin polymer filament length is correlated with sputum cohesivity. Tβ4 depolymerizes CF sputum actin in both a dose-dependent and a time-dependent manner. An apparent synergy of Tβ4 on actin and dornase on DNA may be explained by the combined effect of actin depolymerization and DNA filament severing or by virtue of actin depolymerization increasing the effectiveness of dornase alfa.