Enteric coated gelatin capsule device containing guar gum (GG) microspheres loaded with 5-fluorouracil (5-FU) were developed. 5-FU loaded GG microspheres prepared by water-in-oil (w/o) emulsification method and loading of 5-FU done by both soaking and in-situ techniques during emulsion cross linking procedure. Absence of interaction of 5-FU with polymeric matrix and the cross linking agent was confirmed by Fourier transform infrared (FTIR) spectral study. Thermal studies confirmed the stability and molecular level dispersion of 5-FU in polymer matrix. Swelling experiments revealed that extended cross-linking led to formation of more rigid microspheres and reduction of swelling behavior. In the present study 5-FU loading was 11.28, 3.55, 12.25 and 4.62% with respect to GG microspheres at 1.5 h and 3 h with soaking and in-situ technique respectively. Enteric coated pulsatile capsule device of 5-FU loaded guar gum microspheres were developed and further, in-vitro release studies were conducted at pH 1.2 and pH 7.4 to simulate actual GSF (gastrointestinal fluid) and GIT (gastrointestinal tract) condition respectively. The results indicated that enteric coated capsule device significantly lowers the initial burst effect when compared to microspheres formulation. Colon-targeted release of enteric coated capsule device was achieved successfully over the time period of 2–8 h.