Defective airway mucus clearance is a defining characteristic of CF lung disease, and improvements to current mucolytic strategies are needed. Novel approaches targeting a range of contributing mechanisms are in various stages of preclinical and clinical development. ARINA-1 is a new, nebulized product comprised of ascorbic acid, glutathione, and bicarbonate. Using micro-optical coherence tomography, we tested the effect of ARINA-1 on central features of mucociliary clearance in F508del/F508del primary human bronchial epithelial cells to assess its potential as a mucoactive therapy in CF. We found that ARINA-1 significantly augmented mucociliary transport (MCT) rates, both alone and with CFTR modulator therapy, whereas airway hydration and ciliary beating were largely unchanged compared to PBS vehicle control. Analysis of mucus reflectivity and particle tracking microrheology indicated that ARINA-1 restores mucus clearance by principally reducing mucus layer viscosity. The combination of bicarbonate and glutathione elicited increases to MCT rate comparable to those seen with ARINA-1, indicating the importance of this interaction to the impact of ARINA-1 on mucus transport; this effect was not recapitulated with bicarbonate alone or bicarbonate combined with ascorbic acid. Assessment of CFTR chloride transport revealed an increase in CFTR-mediated chloride secretion in response to ARINA-1 in CFBE41o- cells expressing wild-type CFTR, driven by CFTR activity stimulation by ascorbate. This response was absent in CFBE41o- F508del cells treated with VX-809 and primary HBE cells, implicating CFTR-independent mechanisms for the effect of ARINA-1 on CF mucus. Together, these studies indicate that ARINA-1 is a novel potential therapy for the treatment of impaired mucus clearance in CF.