Colon-specific drug delivery systems (CDDS) can improve the bioavailability of drug through the oral route. A novel formulation for oral administration using pH-enzyme Di-dependent chitosan mcirospheres (MS) and 5-Fu as a model drug has been investigated for colon-specific drug delivery by the emulsification/chemical cross-linking and coating technique, respectively. The influence of polymer concentration, ratio of drug to polymer, the amount of crosslinking agent and the stirring speed on the encapsulation efficiency, particle size in microspheres were evaluated. The best formulation was optimized by an orthogonal design. Drug release studies under conditions mimicking stomach to colon transit have shown that the drug was protected from being released in the physiological environment of the stomach and small intestine. The plasma concentrations of 5-Fu after oral administration of coated chitosan MS to rats were determined and compared with that of 5-Fu solution. The in vivo pharmacokinetics study of 5-Fu loaded pH-enzyme Di-dependent chitosan MS showed sustained plasma 5-Fu concentration-time profile. The in vitro release correlated well with the pharmacokinetics profile. The results clearly demonstrated that the pH-enzyme Di-dependent chitosan MS is potential system for colon-specific drug delivery of 5-Fu.