Heat shock protein 27 (HSP27) is an intracellular stress protein with the cytoprotective effect for a variety of noxious stresses. In this study, using a protein delivery system, we demonstrated the potential cytoprotective effect of HSP27 as a therapeutic protein in cardiac cells and ischemia/reperfusion animal model. We constructed a recombinant HSP27 fused to the protein transduction domain (PTD) derived from HIV-1 TAT protein. Purified recombinant TAT-HSP27 protein was efficiently delivered to H9c2 cells, and its transduction showed cytoprotective effect against the hypoxic stress. Moreover, transduction of TAT-HSP27 also attenuated hypoxia-induced apoptosis, which was accompanied by reduced caspase-3 activity. In addition, intraperitoneal injection of TAT-HSP27 into rat resulted in efficient protein transduction in heart tissues, decreased infarcted myocardium (control vs TAT-HSP27, 39.1% vs 29.5%, P<0.05) and preserved heart function (fractional shortening, 15.6% vs 33.4%, P<0.05), as determined at 7 d after I/R. These results suggest that the PTD-mediated delivery of HSP27 protein may represent a potential therapeutic strategy as protein drug for ischemic heart diseases.