Insulin (Ins) spontaneously and easily complexed with the hydrogel nanoparticle of hydrophobized cholesterol-bearing pullulan (CHP) in water. The complexed nanoparticles (diameter 20-30 nm) thus obtained formed a very stable colloid. The thermal denaturation and subsequent aggregation of Ins were effectively suppressed upon complexation. The complexed Ins was significantly protected from enzymatic degradation. Spontaneous dissociation of Ins from the complex was barely observed, except in the presence of bovine serum albumin. The original physiological activity of complexed Ins was preserved in vivo after i.v. injection.