Abstract: AIM To prepare oral colon-specific metronidazole microsphere delivery system. METHODS Metronidazole microspheres were prepared with Quasi-emulsion method, and the colon site-specific delivery prescription and preparation optimized with fractional factorial design. RESULTS The drug loading of colon site-specific delivery metronidazole microspheres was (39.94±0.15)%, and release fraction 71.78% in an artificial colon solution. CONCLUSION Optimized by fractional factorial design metronidazole microspheres possess the characteristics of a colon site-specific delivery system that delivers its most part in an artificial solution.