An in situ thermogelling, mucoadhesive formulation based on N-trimethyl chitosan chloride has been evaluated for its potential to affect the transmucosal delivery of insulin via the nasal route. In vitro studies at a physiologically relevant temperature (ca. 35 °C) have shown that the formulation releases most of its insulin load (ca. 70%) in a non-Fickian manner during the timescale over which the sol-to-gel transition (ca. 8 min) takes place, and also that, once gelation is complete, the release of the remainder of the therapeutic content follows first order kinetics over at least sixty minutes. Investigations on the effects of the application of the same formulation to a modelled nasal mucosa (Calu-3 cell monolayer) have indicated the capability of the formulation to induce the transient opening of tight junctions. Cytotoxic investigations have shown that the formulation exhibits negligible detrimental effects to the integrity of these monolayers. The in vivo potential of the nasal formulation to act as a once-a-day dosage form for the intranasal delivery of insulin has been demonstrated in a diabetic-rat model.