In recent years, oral colon specific drug delivery system has been paid more attention in the treatment of inflammatory bowel disease (IBD). As the special pH condition in gastrointestinal tract, the challenge for treatment of IBD was that the colon drug delivery system should endure the low pH in stomach and release drugs quickly in high pH in colon. Icariin with the poor solubility and low bioavailability limited the treatment of many diseases in clinic. In this study, the protective mechanism of alginate-chitosan microspheres loaded with icariin were investigated with trinitrobenzene sulfonic acid (TNBS)/ethanol induced colonic mucosal injury in rats. The results of drug release showed that the icariin loaded into microspheres released only 10% in simulated gastric fluid and a high amount of 65.6% released in simulated colonic fluid. The fluorescence tracer indicated high retention of targeted microspheres more than 12h in colon. The microspheres loaded with icariin could not only reduce the colonic injury by decreasing the colon mucosa damage index in rats, but also reduce the inflammatory response by reducing the production and gene expression of inflammatory mediators and cytokines in colonic mucosa. All the results indicate that targeted microspheres loaded with icariin could exert the colon-protective effects through reducing the inflammatory response, which would be developed as a potential drug controlled release system for treatment of ulcerative colitis.