The aim of this study is to elucidate the impact of surface charge of glycerol monooleate-based nanoparticles (NPs) on the cellular uptake and its distribution in the cochlea. These NPs are modified using varied concentration of anionic or cationic lipid. Upon dilution, these lipid mixtures self-assemble to form a series of cubic NPs with various surface charges, but with similar particle size. Positively charged NPs exhibited dose-dependent cytotoxicities against L929 cells proportional to the concentration of cationic lipid; whereas negatively charged NPs did not show obvious cytotoxic properties as compared to unmodified NPs. Meanwhile, confocal microscopy and flow cytometry results suggested that NPs with high positive surface charge were taken up more efficiently by L929 cells. The permeability of round window membrane (RWM) was high for highly positively charged NPs, which is likely due to their highly cellular uptake efficiency and consequently high concentration gradient between RWM and cochlear fluid. More importantly, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) modified NPs greatly facilitated broadly distribution in cochlea, favoring the treatment of hearing loss of low frequencies. Taken together, these findings about charge-dependent of NPs on RWM permeability and cochlear distribution could serve as guideline in the rational design of NP for drug and gene delivery to inner ear.