Positively and negatively charged chitosan derivatives can be used to obtain nanoparticles with different properties. The aim of this work was to determine the role of charge, hydrophobicity and solubility at neutral pH in CH cell penetration and cytotoxicity. We showed that positively charged unmodified CH does not penetrate the cells and is not toxic. Quaternization of CH (CHq) led to the increase in solubility, cell penetration, and cytotoxicity manifested by reactive oxygen species production, cell cycle arrest and inhibition of cell proliferation. Modification of CHq with hydrophobic residues significantly diminished cytotoxic activity however preserved cell penetration. Negatively charged CH derivative succinyl chitosan (CHs) penetrated the cells highly efficiently and demonstrated the stimulation of cell proliferation. Thus, quaternized hydrophobic CH derivatives can be recommended to develop positively charged delivery systems, while any negatively charged chitosans are safe to design nanoparticles.