Mucus obstruction of the airway in patients with cystic fibrosis (CF) reduces lung function, invites infection and limits delivery of inhaled drugs including gene therapy vectors to target cells. Not all patients respond to currently available mucolytics and new approaches are needed. Our objectives were to investigate the in vitro effects of unfractionated heparin (UFH) on the morphology and rheology of sputum, and the effect of UFH on diffusion of 200 nm nanospheres through sputum from adult CF patients. Confocal laser scanning microscopy (CLSM) was used to image fluorescently stained actin and DNA components of CF sputum, and atomic force microscopy (AFM) was used to image isolated DNA networks. The viscoelasticity of CF sputum was measured using dynamic oscillatory rheometry. Nanosphere diffusion was measured through CF sputum using a Boyden chamber-based assay. Actin/DNA bundles in CF sputum were disaggregated by UFH at concentrations of 0.1-10 mg/ml and UFH enhanced the endonuclease activity in sputum from patients on dornase alfa therapy. UFH significantly reduced the elasticity and yield stress, but not the viscosity, of CF sputum from patients not receiving dornase alfa therapy. Heparin dose-dependently significantly increased the diffusion of nanospheres through CF sputum from patients not on dornase alfa therapy from 10.5±2.5 % at baseline to 36.9±4.4 % at 10 mg/ml, but was more potent, with maximal effect at 0.1 mg/ml, in patients who were on dornase alfa therapy. Thus the mucoactive properties of UFH indicate its potential as a new therapeutic approach in patients with cystic fibrosis.