Chitosan is employed as an absorption enhancer for drug delivery strategies. Aim of this study was to investigate the rapid effects on barrier properties of the intestinal epithelial cell model HT-29/B6. Chitosan (0.005%) induced a fast decrease in transepithelial resistance (R^t) which was completely reversible after wash-out. Two-path impedance spectroscopy revealed that chitosan affects both, the paracellular (R^para) and the transcellular (R^trans) resistance. pH-dependence and inhibition of both effects by negatively charged heparin indicated a chitosan action only in the protonated form. The decrease in R^trans was mediated by activation of a chloride-bicarbonate exchanger involved in intracellular pH regulation. This activation was coupled to the decrease in R^para which was associated with an increase in ion permeability and permeability for paracellular flux markers up to 10 kDa. No effects on expression and subcellular distribution of tight junction (TJ) proteins or the actin cytoskeleton were observed. Accordingly, inhibition of actin–myosin interaction, Ca²⁺-dependent intracellular signaling, PKC, PI3K/Akt, MAP kinase p38, and endocytosis pathways did not impair the chitosan effect. These results suggest that the rapid and reversible absorption-enhancing chitosan effect is due to changes in intracellular pH caused by the activation of a chloride-bicarbonate exchanger resulting in the opening of the TJ.