Polycationic compounds, such as poly-L-arginine and poly-L-ornithine (PLO), enhance the nasal absorption of hydrophilic macromolecular drugs. However, the bio availability corresponding to the dose of these enhancers has not been obtained in an open system study, where an administered solution is transferred to the pharynx because they do not exhibit mucoadhesion/retention in the nasal cavity. In this study, we prepared PEGylated-poly-L-ornithine (PEG-PLO) and investigated the effects of PEGylation on in vitro adhesion/retention properties, permeation enhancement efficiency, and cytotoxicity. PEG-PLO bearing 3-4 polyethylene glycol (PEG) chains per PLO molecule was more retentive than unmodified PLO on an inclined plate. The permeability of a model drug, FD-4, across Caco-2 cell sheets was enhanced by PEG-PLO as well as by PLO. PLO showed cytotoxicity at high concentrations, whereas PEG-PLO did not decrease cell viability, even above the concentration giving a sufficient enhancement effect. These findings suggest that PEGylation of polycationic absorption enhancers improves their adhesion/retention and decreases their cytotoxicity, which may lead to enhancers with greater utility.