Polysaccharide nanogel-crosslinked hydrogels for sustained protein delivery systems were prepared using acryloyl group-modified cholesterol-bearing pullulan (CHPOA) as a building block and multiarmed poly(ethylene glycol) (PEGSH) with thiol groups as a cross-linker. Immobilized nanogels were released by the cleavage of ester bonds between the nanogels and cross-linkers according to environmental factors such as pH and the presence of serum. Fluorescein-isothiocyanate-labeled insulin (FITC-Ins)-containing hydrogel was prepared by the cross-linking of FITC-Ins-complexed nanogel and PEGSH. The protein-complexed nanogels were gradually released from the hydrogel by hydrolysis, and then the proteins in the nanogel were released by exchange reactions in the presence of bovine serum albumin. The CHPOA-PEGSH hydrogel is useful as a sustained protein delivery system.