The aim of this study was to evaluate the impact of in situ cross-linkers on the gelling and mucoadhesive properties of thiomers. Polycarbophil-cysteine conjugate (PCP-cys) was synthesized by covalent attachment of l-cysteine to polycarbophil via amide bond formation mediated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and N-hydroxysuccinimide (NHS) whereas in situ cross-linkers (PAA-cys-MNA) were synthesized by the same bond formation between poly(acrylic acid) (PAA) of 2.1-, 6-, and 15 kDa and 2-((2-amino-2-carboxyethyl)disulfanyl)nicotinic acid (cys-MNA) used as ligand. The in situ cross-linking properties were studied via rheological measurements of dynamic viscosity of mixtures of PCP-cys and PAA-cys-MNA with purified porcine intestinal mucus and via rotating cylinder method. The diffusion of polymers in purified porcine intestinal mucus was studied via rotating tube technique. The results showed that in situ cross-linkers (PAA 2.1-, 6-, 15 kDa) increase the dynamic viscosity of PCP-cys/mucus mixtures by 5.1-, 5.6-, and 3.5-fold. Combinations of 10% of in situ cross-linkers PAA 2.1-, 6- or 15 kDa and 90% PCP-cys increased the adhesion time 1.1-, 2.0- and 4.9-fold, respectively, compared to PCP-cys alone. Diffusion study showed that low molecular mass PAAs highly penetrate into the mucus gel layer due to their high polymer chain mobility compared to PCP-cys. The results provide evidence for the potential of in situ cross-linking agents as gelling and mucoadhesion enhancers.