In the present study, chitosan (CS) was thiolated by introducing L-cysteine via amide bond formation. Free thiol groups were protected with highly reactive 6-mercaptonicotinic acid (6-MNA) and less-reactive L-cysteine, respectively, via thiol/disulfide-exchange reactions. Unmodified CS, L-cysteine-modified thiolated CS (CS-Cys), 6-MNA-S-protected thiolated CS (CS-Cys-MNA), and L-cysteine-S-protected thiolated CS (CS-Cys-Cys) were applied as coating materials to solid lipid nanoparticles (SLN). The strength of mucus interaction followed the rank order plain < CS < CS-Cys-Cys < CS-Cys < CS-Cys-MNA, whereas mucus diffusion followed the rank order CS-Cys < CS-Cys-Cys < CS < CS-Cys-MNA < plain. In accordance with lower reactivity, CS-Cys-Cys-coated SLN were immobilized to a lower extent than CS-Cys-coated SLN, while CS-Cys-MNA-coated SLN dissociated from their coating material resulting in a similar diffusion behavior as plain SLN. Consequently, CS-Cys-Cys-coated SLN and CS-Cys-MNA-coated SLN showed the highest retention on porcine intestinal mucosa by enabling a synergism of efficient mucus diffusion and strong mucoadhesion.