Aim Calpain activation has a putative role in ischaemia‐reperfusion injury of cardiomyocytes. This study clarified the in vivo contribution of calpain to disruption of cardiomyocyte sarcolemma during ischaemia and after reperfusion in anaesthetized rats. Methods Using a microdialysis technique in the hearts of anaesthetized rats, we investigated the effects of the calpain inhibitors on myocardial interstitial myoglobin level in the ischaemic region during coronary occlusion and after reperfusion. The calpain inhibitors were administered locally via a dialysis probe. Two durations of coronary occlusion were tested. Results Thirty‐minute coronary occlusion: dialysate myoglobin concentration increased markedly from 385 ± 46 ng mL−1 at baseline to 3701 ± 527 ng mL−1 at 20–30 min of occlusion. After reperfusion, dialysate myoglobin concentration further increased, reaching a peak (12 296 ± 1564 ng mL−1) at 10–20 min post‐reperfusion and then declined gradually. The calpain inhibitors, MDL‐28170 and SNJ‐1945 did not change dialysate myoglobin concentration during occlusion but attenuated the increase after reperfusion to 6826 ± 1227 and 8130 ± 938 ng mL−1 at 10–20 min post‐reperfusion (P < 0.05), respectively. Ninety‐minute coronary occlusion: dialysate myoglobin concentration increased from 516 ± 33 ng mL−1 at baseline to 5463 ± 387 ng mL−1 at 80–90 min after occlusion. After reperfusion, there was no significant increase in dialysate myoglobin concentration. MDL‐28170 did not affect dialysate myoglobin concentration during occlusion or after reperfusion. Conclusion Calpain contributes to sarcolemmal disruption immediately after reperfusion following 30‐min coronary occlusion, but has little effects during ischaemia and after reperfusion in 90‐min coronary occlusion.