For the delivery of bioactive components to the colon, an oral colon-specific controlled release system coated with a resistant starch-based film through aqueous dispersion coating process was developed. Starch was modified by a high-temperature-pressure reaction, enzymatic debranching, and retrogradation, resulting in a dramatic increase in the resistibility against enzymatic digestion (meaning the formation of resistant starch, specifically RS3). This increase could be associated with an increase in the relative crystallinity, a greater amount of starch molecular aggregation structure, and the formation of a compact mass fractal structure, resulting from the treatment. The microparticles coated with this RS3 film showed an excellent controlled release property. In streptozotocin (STZ)-induced type II diabetic rats, the RS3 film-coated insulin-loaded microparticles exhibited the ability to steadily decrease the plasma glucose level initially and then maintain the plasma glucose level within the normal range for total 14-22 h with different insulin dosages after oral administration; no glycopenia or glycemic fluctuation was observed. Therefore, the potential of this new RS3 film-coated microparticle system has been demonstrated for the accurate delivery of bioactive polypeptides or protein to the colon.