Sperminated pullulans (SP) having different molecular weights (MWs) were prepared, and the enhancing effect on the pulmonary absorption of insulin in rats was examined. SP acted as enhancers of insulin absorption when a 0.1% solution was applied with insulin simultaneously and their enhancing effects depended on the MW of the SP; the same solutions exhibited low toxicity in the in vivo LDH leaching test. In the in vitro experiments using Calu-3 cells, tight junction-opening effects and a toxic effect of SP in the MTT assay were observed at lower concentrations compared with the in vivo experiments. A mucus layer might interfere with the interaction between SP and the cell surface and might suppress both these effects and toxicity. SP having a high MW will be useful for preparing safe and efficient formulations of peptide and protein drugs. The change in the localization of the tight junction proteins may be related to the permeation-enhancing mechanism of SP.