The aim of this paper was to evaluate the penetration enhancement properties of nanoparticles (NP) based on N-trimethyl chitosan (TMC 35% quaternization degree) loaded with insulin. The permeation performances of TMC NP were compared with those of chitosan (CS) NP and also with TMC and CS solutions. To estimate the mechanism of penetration enhancement, two different approaches have been taken into account: an in vitro study (Caco-2 cells) and an ex vivo study (excised rat duodenum, jejunum, and ileum). Insulin-loaded CS and TMC NP had dimensions of about 250 nm and had high yield and high encapsulation efficiency. The in vitro study evidenced that TMC and CS were able to enhance insulin permeation to the same extent. Penetration enhancement properties of TMC NP seem to be prevalently related to endocytosis while the widening of tight junctions appeared more important as mechanism in the case of CS NP. The ex vivo study put in evidence the role of mucus layer and of its microclimate pH. In duodenum (pH 5-5.5), CS and TMC solutions were more effective than NP while TMC NP were more efficient towards jejunum tissue (pH 6-6.5) for their high mucoadhesive potential. Confocal laser scanning microscopy study supported the hypothesis that penetration enhancement due to TMC NP was mainly due to internalization/endocytosis into duodenum and jejunum epithelial cells. The good penetration enhancement properties (permeation and penetration/internalization) make TMC NP suitable carriers for oral administration of insulin.