Controlled release delivery system of chemotherapeutic agents at the site of colon endorses modern drug-entrapped delivery tools, which release the entrappedagents at a controlled rate for anextended period providing patient compliance and additional protection from the degradinggastric environment. Thus, the present study was aimed to develop and optimize a novel polymeric microsphere of 5-fluorouracil (5-FU) using natural gum katira to obtain an optimal therapeutic response at the colon. Due course of experimentation, in-vivo safety profile of the gum katira in an animal model was established. Modified solvent extraction/evaporation technique wasemployed to encapsulate 5-FU in the natural polymeric microsphere and was characterized using in-vitro studies to investigate particle size, morphology, encapsulation efficiency and release of the drug from developed formulation. Formulated and optimized polymeric microsphere of 5-FU using gum katira polymer own optimal physicochemical characteristics with a fine spherical particle with size ranged from 210.37±7.50 to 314.45±7.80 μm.Targeted microsphere exhibited good cytotoxicity and also has high drug entrapment efficiency, and satisfactory release pattern of the drug within a time frame of 12 h. Finally, we foresee that the optimized polymeric gum katiramicrosphere of 5-FU could be a promising micro-carrier for efficient colon drug targeting delivery tool with improved chemotherapeutic efficacy against colon cancer.