Polyethylene glycol cross-linked chitosan microspheres loaded with 5-fluorouracil (5-FU) have been prepared by emulsion cross-linking and enteric coated with cellulose acetate phthalate (CAP) to facilitate direct targeting of 5-FU to the colon. Spherical microspheres with surface ridges to moderate smooth surfaces loaded with different concentrations (i.e., 10, 20, and 30% w/w) of 5-FU have been evaluated for encapsulation efficiency, swelling and in vitro release characteristics in simulated gastro intestinal tract (GIT) conditions for both enteric coated and uncoated microspheres. In acidic pH, 8 to 17%, but in alkaline pH, approximately 88 to 97% of 5-FU was released with CAP coated microspheres. In the case of uncoated microspheres, 46 to 77% release of 5-FU occurred in acidic pH, but 93 to 97% was released in alkaline pH. The study demonstrates that chitosan coating with CAP and cross-linking with PEG is necessary for targeted delivery of 5-FU to the colon. The coated microspheres are found to be more suitable for colon targeting than the uncoated formulations as the former prolonged the 5-FU release from 6 to 12 h by protecting 5-FU in acidic environment of the stomach. Kinetics of drug release as investigated by empirical equations suggested Super Case II transport mechanism.