The effects of five chitosan oligomers on the intestinal absorption of fluorescein isothiocyanate-labeled dextrans (FDs) and insulin were studied by an in situ loop method. The absorption of FD4 from the jejunum was effectively improved in the presence of 0.5% (w/v) chitosan hexamer and dimer. However, chitosan hexamer did not improve the colonic absorption of FD4, although we found a moderate increase in the colonic absorption of FD4 in the presence of chitosan pentamer and dimer. The absorption enhancing effect of chitosan hexamer decreased as the molecular weights of FDs increased. In addition, we found a remarkable increase in plasma insulin levels and a significant hypoglycemic effect after jejunal administration of insulin with chitosan hexamer. In the toxicity studies of chitosan hexamer, we found no significant increase in the release of total protein and activity of lactate dehydrogenase (LDH) from the intestinal epithelium in the presence of chitosan hexamer (0.5%, w/v), indicating that this compound was a safe absorption enhancer for improving the intestinal absorption of poorly absorbable drugs. Finally, the transepithelial electrical resistance (TEER) and the permeability of FD4 in rat jejunal membranes with or without chitosan hexamer (0.5%, w/v) were examined by an in vitro diffusion chamber method. We observed a moderate decrease in the TEER values of rat jejunal membranes and a corresponding increase in the permeability of FD4 in the presence of chitosan hexamer (0.5%, w/v). These findings suggested that chitosan hexamer might loosen the tight junction of the intestinal epithelium, thereby improving the intestinal permeability of hydrophilic macromolecular compounds via a paracellular pathway.