AIM: Studying the mechanism of protective role of metallothionein (MT) in hypoxic preconditioning(HPC) of cultivated rat cardiomyocytes. METHODS: Using the model of hypoxia/reoxygenation of cultivated rat cardiomyocytes. Determing the contents of MT, malonyldialdehyde (MDA) - metabolism product of lipid peroxidation and the activities of Na+ - K+ ATPase, Ca2+ - Mg2+ ATPase of cardiomyocytes 24 h after HPC, the determining the relevant changes after using MT antibody. RESULTS: After 24 h in HPC, the contents of MT and activities of Na+ - K+ ATPase, Ca2+ - Mg2+ ATPase were obviously higher than those in the control and hypoxia/reoxygenation(P＜ 0. 05 ), and the contents of MDA were decreased remarkedly (P ＜ 0.01 ). Then after using MT antibody, the activities of two enzyme were progressively decreased and the contents of MDA were significanily higher than those in the control and MT antibody - free groups(P ＜ 0.01 ). CONCLUSION: HPC may induce excessive synthesis of MT, and MT can protect myocardial reoxygenation injury by eliminating lipid peroxidation and rising the activities of Na+ - K+ ATPase and Ca2+ - Mg2+ ATPase.