The prime objective of the present study was to develop novel colon targeting xanthan gum/polyvinylpyrrolidone-co-poly acrylic acid hydrogels for controlled delivery of 5-fluorouracil at colon-specific site by combining the properties of natural and synthetic polymers. Xanthan gum (XG) and polyvinylpyrrolidone (PVP) polymer have been chemically cross-linked with acrylic acid (AA) monomer using ethylene glycol dimethacrylate and ammonium per sulfate/sodium hydrogen sulfite as a cross-linker and initiator, respectively. Different proportions of XG, PVP, acrylic acid and ethylene glycol dimethacrylate were blended with each other to fabricate pH-sensitive hydrogels by free radical polymerization. SEM, FTIR, TGA, DSC, XRD and sol-gel fraction analysis were carried out for characterization and structural analysis of polymeric system. pH-responsive behavior of fabricated hydrogels was investigated by performing swelling studies and in vitro drug release studies at both pH 1.2 and pH 7.4. Toxicity studies were performed on rabbits to evaluate cytotoxicity and biocompatibility. TGA and DSC confirmed that formulations were thermodynamically stable, while FTIR, SEM and XRD revealed the successful grafting of components. By increasing the content of polymer, monomer and cross-linker, gel fraction was found to be increased. Swelling capacity of hydrogels increases with the increase in concentration of monomer, while swelling capacity tends to decreases with the increase in concentration of cross-linker and polymer in hydrogel composition. pH sensitivity of hydrogels was confirmed by swelling dynamic and drug release behavior in simulated gastrointestinal fluids. Toxicity studies confirmed that hydrogels were non-toxic. Drug release kinetics revealed the controlled release pattern of 5-fluorouracil in developed polymeric network. Cross-linked XG/PVP-co-poly (AA) hydrogels can be used as promising candidate for controlled delivery of 5-FU for prolonged treatment period at colon-specific site.