Abstract Background Universal demand for cheese keeps the search for appropriate enzymes from plants mimicking animal rennet action in scientific focus. Objective To associate distribution of milk clotting potential and profile of whole/κ-casein hydrolysis by Calotropis gigantea stem, leaf and respective calli crude enzymes (CE). Materials and methods Milk clotting activity and index were assayed for CE. Caseinolytic activity (CA) was evaluated spectrophotometrically. 0.5 CA units of CE and Enzeco ® were used for studying whole/κ-casein hydrolysis pattern by Tricine SDS-PAGE. Inhibition studies were performed for enzyme characterisation. Results Traditionally propagated (TP) stem and its callus CE exhibited high specific milk clotting activity (1297.30±0.2 U/mg of protein and 926.74±44.13 U/mg of protein, respectively) and milk clotting index (103.562±1.162 and 79.365±4.93, respectively). Comparison of whole casein hydrolytic pattern by 0.5 CA units of CE revealed closer resemblance between leaf callus and Enzeco ® . However, κ-casein specificity analysis revealed TP leaf to be closely mimicking the performance by Enzeco ® . Conclusion Study suggests CE from TP leaf to be a potential vegetable coagulant to work as an effective and low-cost milk clotting mediator in cheese industry.