In the present work calcium pectinate (Ca-pectinate) microspheres were prepared to deliver methotrexate in the environment of colon. Calcium pectinate microspheres were prepared by modified emulsification method using calcium chloride as cross linker. All the formulations were evaluated for various physicochemical parameters. Particle size of the microspheres was determined using laser diffraction particle size analyzer. Encapsulation efficiency was determined by digesting with enzyme pectinase for 24 hours and swellability by equilibrium swelling in simulated gastrointestinal fluid. The in vitro drug release studies were performed in simulated gastric fluid for 2 hours and simulated intestinal fluid for 3 hours. In vitro release rate studies were also carried out in simulated colonic fluid in presence of rat caecal contents. Moreover, release rate studies were also carried out after enzyme induction by treating the rats with 1 ml of 1% w/v aqueous dispersion of pectin for 7 days. Mean particle size of the microspheres was found to be in the range of 20.82±1.34 to 32.26±1.59 μm whereas the entrapment efficiency varied from 52.28±0.32 to 74.01±3.32%. The in vitro drug release studies in simulated gastric fluid and simulated intestinal fluid showed that only 8.15±0.49% drug was released in 5 hours whereas most of the loaded drug was released in simulated colonic fluid containing pectinase. In vitro release rate study showed release of 69.94±3.46% of drug in presence of 3% rat caecal contents, which was further increased to 94.43±4.48% when enzyme induction was carried out for 7 days. Thus, it is concluded that calcium pectinate microspheres can be used to effectively localize the release of drug in the physiological environment of colon.