Chitosan-6-mercaptonicotinic acid (chitosan-6-MNA) is a thiolated chitosan with strong mucoadhesive properties and a pH-independent reactivity. This study aimed to evaluate the in vivo potential for the oral delivery of insulin. The comparison of the nonconjugated chitosan and chitosan-6-MNA was performed on several studies such as mucoadhesion, release, and in vivo studies. Thiolated chitosan formulations were both about 80-fold more mucoadhesive compared with unmodified ones. The thiolated chitosan tablets showed a sustained release for 5 h for the polymer of 20 kDa and 8 h for the polymer of 400 kDa. Human insulin was quantified in rats' plasma by means of ELISA specific for human insulin with no cross-reactivity with the endogenous insulin. In vivo results showed thiolation having a tremendous impact on the absorption of insulin. The absolute bioavailabilities were 0.73% for chitosan-6-MNA of 20 kDa and 0.62% for chitosan-6-MNA 400 kDa. The areas under the concentration-time curves (AUC) of chitosan-6-MNA formulations compared with unmodified chitosan were 4.8-fold improved for the polymer of 20 kDa and 21.02-fold improved for the polymer of 400 kDa. The improvement in the AUC with regard to the most promising aliphatic thiomer was up to 6.8-fold. Therefore, chitosan-6-MNA represents a promising excipient for the oral delivery of insulin.