Insulin-containing layer-by-layer (LbL) thin films were prepared by an alternate deposition of insulin and anionic polysaccharides (heparin, kappa-carrageenan, and fucoidan) through an electrostatic force of attraction between positively charged insulin and anionic polysaccharides at pH 3.0. The loading of insulin in the LbL films increased with the increasing number of layers (or the film thickness), depending on the polysaccharide type. LbL films composed of kappa-carrageenan contained higher amount of insulin than in heparin-and fucoidan-based films. The LbL films were fairly stable in acidic media, while insulin was released from the films in weakly acidic and neutral solutions as a result of loss of net positive charge in insulin. The released insulin retained its original structure.