This study determined the effects of glutamate on the Ca2+ paradoxical heart, which is a model for Ca2+ overload‐induced injury during myocardial ischaemia and reperfusion, and evaluated its effect on a known mediator of injury, calpain. An isolated rat heart was retrogradely perfused in a Langendorff apparatus. Ca2+ paradox was elicited via perfusion with a Ca2+‐free Krebs‐Henseleit (KH) solution for 3 minutes followed by Ca2+‐containing normal KH solution for 30 minutes. The Ca2+ paradoxical heart exhibited almost no viable tissue on triphenyltetrazolium chloride staining and markedly increased LDH release, caspase‐3 activity, cytosolic cytochrome c content, and apoptotic index. These hearts also displayed significantly increased LVEDP and a disappearance of LVDP. Glutamate (5 and 20 mmol/L) significantly alleviated Ca2+ paradox‐induced injury. In contrast, 20 mmol/L mannitol had no effect on Ca2+ paradox. Ca2+ paradox significantly increased the extent of the translocation of μ‐calpain to the sarcolemmal membrane and the proteolysis of α‐fodrin, which suggests calpain activation. Glutamate also blocked these effects. A non‐selective inhibitor of glutamate transporters, dl‐TBOA (10 μmol/L), had no effect on control hearts, but it reversed glutamate‐induced cardioprotection and reduction in calpain activity. Glutamate treatment significantly increased intracellular glutamate content in the Ca2+ paradoxical heart, which was also blocked by dl‐TBOA. We conclude that glutamate protects the heart against Ca2+ overload‐induced injury via glutamate transporters, and the inhibition of calpain activity is involved in this process.