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The monosubstituted insulin with poly(ethyleneglycol) (PEG, MW about 2200) formed polypseudorotaxanes with αand γ-cyclodextrins (CyDs), by inserting one PEG chain of the pegylated insulin in the α-CyD cavity and two PEG chains in the γ-CyD cavity.The pegylated insulin/a-and γ-CyD polypseudorotaxanes were less soluble in water and the release rate of the drug decreased in the order of drug alone > the γ-CyD polypseudorotaxane > the α-CyD polypseudorotaxane.The subcutaneous administration of the pegylated insulin/γ-CyD polypseudorotaxane in rats significantly sustained plasma glucose levels with an enhanced hypoglycemic effect.The results indicated that the pegylated insulin/CyD polypseudorotaxanes can work as a sustained drug release system.