The effect of guluronate oligomers on the barrier properties of mucous matrices was investigated in terms of the mobility of nanoparticles in mucous matrices by fluorescence recovery after photobleaching (FRAP), cellular uptake of nanoparticles in mucus secreting cells (HT29-MTX), and mucin matrix architecture by scanning electron microscopy (SEM). Guluronate oligomers improved nanoparticle mobility in both native and highly purified mucus matrices and improved cellular uptake of nanoparticles through a mucus layer. Addition of guluronate oligomers to mucin matrices resulted in a decrease in the density of network cross-links and an increase in matrix pore size. Based on these data, we conclude that guluronate oligomers are able to improve nanoparticle mobility in several mucus matrices and alter network architecture in mucin matrices in a manner that suggests a reduction in barrier function. As such, there may be a potential application for guluronate oligomers in mucosal delivery of nanomedicines.