The objective of this study was to develop a novel bacterially-triggered micro-particular system of de-esterified tragacanth (DET) in combination with Eudragit S-100 coated capsules for colon drug delivery of 5-fluorouracil (5-FU) using microemulsion method. The loading study was conducted at different drug-to-polymer ratios and cross-linker concentrations. The maximum loading efficiency was achieved, 44.1% at 1:5 drug-to-polymer ratio and 0.7% cross-linker concentration. The FTIR results also confirmed the encapsulation of 5-FU in microspheres. The release profile was dependent on the cross-linker concentration, environmental pH, and presence of pectinase enzyme. Microspheres inserted into Eudragit S-100 coated capsules released less than 5% of the drug at stomach and small intestine pH levels, whereas 70% of the drug was released at colon pH levels, and about 25% of the drug did not release unless in the presence of pectinase enzyme. To omit burst release, microspheres were washed with water, and the release became pH independent, and was just achieved in the presence of pectinase enzyme. 5-FU loaded microspheres with an IC value of 80 µg/mL were as effective as the free drug on HT-29. Generally, the results demonstrated that drug-loaded microspheres inserted into Eudragit S-100 coated capsules can be effective for colon-targeted delivery.