Stress pretreatments protect myocardium from ischemic injury. We hypothesized that tetracycline, an antibiotic, may induce a stress response via the inhibition of mitochondrial translation as it induces the cold stress response by translational inhibition in E. coli. If so, tetracycline may protect myocardium from ischemic injury as stress pretreatments do. Thus, we investigated the effects of tetracycline on myocardial ischemia and its association with stress response. In a dog model of acute ischemia, 4mg/kg tetracycline injected 30 min prior to the occlusion improved the functional recovery from stunning of myocardium caused by ischemia. The same dosage of tetracycline dramatically reduced the size of infarct area in murine hearts analyzed by tetrazolium staining. In HeLa cells, tetracycline induced molecules that were increased by cold stress, which suggests that tetracycline may induce a cold stress-like response in mammalian cells. These molecules were also induced by ischemic stress in murine hearts, suggesting that the stress response caused by translational inhibition in mitochondria may be associated with the cardioprotection by tetracycline. Our results suggest that a subclinical dosage of tetracycline may protect heart from ischemic injury. Therefore, tetracycline may be of great use in suppressing the development of infarction caused by myocardial ischemia. This study is also important for providing new insights into the non-antimicrobial effects of tetracycline and its derivatives.