Chitosan has a wide range of applications in biomedical materials as well as in dietary supplements. Chitosan oligosaccharide with free-amine group (COFa) is an improvement over traditional chitosan that lacks the usual impurities and materials detrimental to the body. Based on a previous study of water soluble chitosan (WSC, chitosan lactate), we investigated the molecular weight (Mw) - dependent absorption phenomena of COFa in vitro and in vivo with various Mws. The absorption of CO Fa was significantly influenced by its molecular weight. As Mw increases, the absorption decreases. The absorption profiles for 5 K COFa (Mw=5 kDa) were observed to be more than 10 times higher than those of high molecular weight chitosan (100 K HWSC Mw=100 kDa) in both in vitro and in vivo transport experiments. Furthermore, the in vitro transport experiment suggested that transcellular transport of the COFa (Mw <10 kDa) through Caco-2 cell layer could occur with a negligible cytotoxic effect. The COFas showed a cytotoxic effect on Caco-2 cells that was dependent on dose and Mw. COFa could be transported transcellularly through the Caco-2 cell layer.