Multi-functionalized nanoparticles (NPs) have recently been extensively explored for their potential in novel drug delivery and nanomedicine applications. Functionalized NPs based local drug delivery across the mucosal epithelia has gained much interest.Despite reports confirming cellular nanotoxicity effects, possible health hazards resulted from mucus rheological disturbances induced by NPs are underexplored.Accumulation of viscous, poorly dispersed and less transportable mucus that could result in improper mucus rheology and dysfunctional mucociliary clearance are typically found to associate with many respiratory diseases such as asthma, cystic fibrosis (CF) and COPD (chronic obstructive pulmonary disease). Whether functionalized NPs can alter mucus rheology and its operational mechanisms are not resolved. Here we show for the first time that positively-charged functionalized NPs can effectively induce mucin aggregation and hinder mucin gel hydration. These NPs significantly increase the size of aggregated mucin approximately 30 times within 24 hrs. EGTA (ethylene glycol tetraacetic acid, 2 mM) chelation of indigenous mucin crosslinkers (Ca2+ ions) was unable to effectively disperse NPs-induced aggregated mucins. We also found that positively-charged NPs can significantly reduce the swelling kinetics and hydration of newly released mucus. Our results have demonstrated that positively charged functionalized NPs can serve as effective crosslinkers hindering mucin disaggregation and dispersion resulting in potential dysfunctional mucociliary clearance and health problems. This report also highlights the unexpected health risk of NP-induced change in mucus rheology and possible mucociliary transport impairment on epithelial mucosa. In addition, our data can serve as a prospective guideline for designing nanocarriers specific for mucosal epithelia drug delivery applications.(Supported by NSF CBET-0932404, NIH 1R15HL095039-01A1 and CITRIS #31 seed grant)