In cystic fibrosis (CF) lungs, epithelial Na+ channel (ENaC) hyperactivity causes a reduction in airway surface liquid (ASL) volume, leading to decreased mucocilliary clearance and lung damage. Inhibition of ENaC is an attractive therapeutic option. However, ENaC antagonists have failed clinically due to off-target renal effects. The S18 peptide is a naturally occurring short palate lung and nasal epithelial clone 1 (SPLUNC1)-derived ENaC antagonist that restores ASL height for up to 24 h in CF human bronchial epithelial cultures. However, its efficacy and safety in vivo are unknown. To interrogate the potential clinical efficacy of S18, we assessed its safety and efficacy using airway cultures and animal models. S18-mucus interactions were tested using super resolution microscopy, quartz crystal microbalance with dissipation and confocal microscopy. Human and murine airway cultures were used to measure ASL height. Off-target effects were assessed in conscious mice and anesthetized rats. Morbidity and mortality were assessed in the βENaC-Tg mouse model. Restoration of normal mucus clearance was measured in CFTR(inh)-172 challenged sheep. We found that S18 does not interact with mucus and rapidly penetrated dehydrated CF mucus. Compared to amiloride, an early generation ENaC antagonist, S18 displayed a superior ability to slow ASL absorption, reverse CFTR(inh)-172-induced reduction of mucus transport and reduce morbidity and mortality in the βENaC-Tg mouse, all without inducing any detectable signs of renal toxicity. These data suggest that S18 is the first naturally occurring ENaC antagonist to show improved preclinical efficacy in animal models of CF with no signs of renal toxicity.