<p>The objective of this study was to investigate localization and activation of calpain-1 in chicken muscles postmortem and its contribution to meat tenderization. Results showed that plasma membrane bound and sarcoplasmic calpain-1 content increased early postmortem and declined at 24&#xa0;h, while myofibril bound calpain-1 displayed a reversed trend in muscles, indicating the transfer of calpain-1 from sarcolemma and sarcoplasm to myofibrils. Ca²⁺ treatment accelerated the decline of membrane associated calpain-1 and the activation of calpain-1. The translocation was accompanied by remarkable reduction of desmin content and shear force, suggesting that membrane localization facilitated calpain-1 activation and placed the enzyme in proximity to its substrates. The changes of calpain-1 were highly correlated with alterations in Ca²⁺ ATPase activity, Na⁺/K⁺ ATPase activity and mitochondria stability, demonstrating that Ca²⁺ overload might trigger calpain redistribution, membrane damage, and mitochondrial dysfunction, and ultimately influence the tenderness of meat.</p> Graphical Abstract <p></p>