Aim To investigate the mechanism of intracellular calcium overload of endoxin mediating myocardial ischemia reperfusion(MIR) injury, the changes of endoxin level, ATPase activities, intramitochondrial Ca²⁺ concentration and gene expression of Na+-K+-ATPase isoforms in myocardium of rats with MIR were observed. Methods Thirty-two male Sprauge Dawley rats were randomly divided into 4 groups. Sham operation group (control group): silk suture threading the left anterior descending coronary artery without ligature; MIR group (MIR):left anterior descending coronary artery was subjected to 30 min ligation followed by 45 min reperfusion; normal saline group (NS):MIR model was given 5 mL/kg-1 normal saline; verapamil group (Ver): MIR model was given 5 mg/kg verapamil. Drug and NS were injected into vessel via femoral vein within 5 min before reperfusion. After reperfusion left ventricle myocardium samples were processed immediately in order to measure the activities of Na+-K+-ATPase and Ca²⁺-Mg2+-ATPase, endoxin level, intramitochondrial Ca²⁺ concentration and the changes of mRNA and protein levels of α₁, α₂, α3 and β1 isoforms of Na+-K+-ATPase were measured by RT-PCR and western-blot technology respectively. Results After MIR, the level of endoxin in myocardium was obviously increased, the activities of Na+-K+-ATPase and Ca²⁺-Mg2+-ATPase in myocardial membrane were significantly decreased while the concentration of intramitochondrial Ca²⁺ increased, the levels of mRNA and protein of the α₁, α₂, α3 and β1 isoforms of Na+-K+-ATPase were reduced markedly. Verapamil had only effect of reducing the concentration of intramitochondrial Ca²⁺. Conclusion MIR resulted in increase of myocardial endoxin secretion. The latter could depress the activity of Na+-K+-ATPase by changing the gene expression of α₁, α₂, α3 and β1 isoforms of Na+-K+-ATPase in myocardial membrane, and induce intramitochondrial Ca²⁺ overload, thereby mediate MIR injury.