Abstract: Calpastatin, a specific calpain inhibitor was purified to electrophoretical homogeneity from grass prawn (Penaeus monodon) muscle by 100 °C heat-treatment, DEAE-Sephacel, and Q-Sepharose chromatographs. No significant change in the inhibitory activity of crude calpastatin was observed even after 20 min incubation at 100 °C, pH 7.0. The purified prawn calpastatin had a molecular weight (Mr) of 80 and 88.7 kDa determined by SDS−PAGE and Sephacryl S-200 HR gel filtration, respectively. According to the active site titration, the purified calpastatin revealed four beef μ-calpain and two beef m-calpain binding domains, respectively. It was stable during 1 h of incubation at 30 °C under pH 4.5−10.0 and shown to be a highly specific inhibitor for calpain.