Abstract: The present investigation is envisaged to study the production of alkaline and thermostable polygalacturonase using isolated soil Bacillus subtilis (C4) by submerged fermentation. The extracted enzyme was subjected to partial purification which included ammonium sulphate precipitation, dialysis, ion exchange chromatography and affinity chromatography. The cell free supernatant polygalacturonase activity was found to be in the 30-40% salt saturation fraction. Specific activity of polygalacturonase increased from 33.68 to 93.05 U mL-1. Further the fraction was loaded onto anion exchanger, DEAE Sephacel equilibrated with 20mM Tris HCl (pH 7.5 buffer). The proteins were eluted with NaCl gradient. 9.84 fold purification of the enzyme was achieved and its specific activity was found to be 915.6 U mL-1. When subjected to affinity chromatography the purification fold increased up to 15 fold with specific activity 13789.11 U mL-1. The homogeneity of the polygalacturonase was judged by SDS-PAGE. It was found that PGs had a molecular weight ranging between 43 and 66 kDa. The partially purified enzyme was characterized; the enzyme was stable at 60°C up to 60 min at pH 9.0. The enzyme was showing 100% substrate specificity to polygalacturonic acid. A slight activation effect was observed in the presence of Ca2+ and Mg2+.