An intracellular glutaminase-free l-asparaginase from Pectobacterium carotovorum MTCC 1428 was isolated to apparent homogeneity. The homotetramer enzyme has a molecular mass of 144.4kDa (MALDI-TOF MS) and an isoelectric point of approximately 8.4. The enzyme is very specific for its natural substrate, l-asparagine. The activity of l-asparaginase is activated by mono cations and various effectors including Na + , K + , l-cystine, l-histidine, glutathione and 2-mercaptoethanol whereas it is moderately inhibited by various divalent cations and thiol group blocking reagents. Kinetic parameters, K m , V max and k cat of purified l-asparaginase from P. carotovorum MTCC 1428 were found to be 0.657mM, 4.45U μg −1 and 2.751×10 3 s −1 , respectively. Optimum pH of purified l-asparaginase for the hydrolysis of l-asparagine was in the range of 8.0–10.0, and its optimum temperature was found to be 40°C. The purified l-asparaginase has no partial glutaminase activity, which can reduce the possibility of side effects during the course of anti-cancer therapy.