Background Mucin disulfide cross-links mediate pathologic mucus formation in muco-obstructive lung diseases. MUC-031, a novel thiol-modified carbohydrate compound, cleaves disulfides to cause mucolysis. The aim of this study was to determine the mucolytic and therapeutic effects of MUC-031 in sputum from patients with cystic fibrosis (CF) and mice with muco-obstructive lung disease (βENaC-Tg mice). Methods We compared the mucolytic efficacy of MUC-031 and existing mucolytics (N-acetyl cysteine [NAC] and rhDNase) using rheology to measure the elastic modulus (G’) of CF sputum, and we tested effects of MUC-031 on airway mucus plugging, inflammation and survival in βENaC-Tg mice to determine its mucolytic efficacy in vivo. Results In CF sputum, compared to the effects of rhDNase and NAC, MUC-031 caused a larger decrease in sputum G’, was faster in decreasing sputum G’ by 50%, and caused mucolysis of a larger proportion of sputum samples within 15 min of drug addition. Compared to vehicle control, three treatments with MUC-031 in one day in adult βENaC-Tg mice decreased airway mucus content (16.8±3.2 versus 7.5±1.2 nl·mm−2, p<0.01) and bronchoalveolar lavage cells (73,833±6,930 versus 47,679±7736 cells·mL−1, p<0.05). Twice daily treatment with MUC-031 for two weeks also caused decreases in these outcomes in adult and neonatal βENaC-Tg mice and reduced mortality from 37% in vehicle-treated to 21% in MUC-031 treated in βENaC-Tg neonates (p<0.05). Conclusion MUC-031 is a potent and fast acting mucolytic that decreases airway mucus plugging, lessens airway inflammation, and improves survival in βENaC-Tg mice. These data provide rationale for human trials of MUC-031 in muco-obstructive lung diseases.