Highly viscoelastic and adhesive sputum has precluded efficient nanoparticle-based drug and gene delivery to the lungs of patients with cystic fibrosis (CF). We sought to determine whether nanoparticles coated with non-mucoadhesive polymers could penetrate CF sputum, and to use these “muco-inert particles” (MIPs) as non-destructive nanoprobes to characterize the sputum microstructure. Particles as large as 200nm in diameter that were densely coated with low MW polyethylene glycol (PEG) moved through undiluted CF sputum with average speeds up to 90-fold faster than similarly-sized uncoated particles. On the other hand, the transport of both coated and uncoated 500nm particles was strongly hindered. The local viscosity of sputum, encountered by the fastest 10% of 200nm MIPs, was only 5-fold higher than that of water, whereas the bulk viscosity was 10,000-fold higher at low shear rates. Using measured transport rates of various sized MIPs combined with an obstruction-scaling model, we determined that the average 3D mesh spacing of CF sputum is ∼140±50nm (range: 60–300nm). Taken together, these results demonstrate that nanoparticles up to 200nm in diameter that do not adhere to CF sputum can move rapidly through this critical barrier by accessing pores that are filled with a low viscosity fluid. The results also offer hope that desperately needed sputum-penetrating drug- and gene-carrier nanoparticles can be developed for CF.