Pathophysiological hypoxia is an important modulator of gene expression in cardiomyocytes. Recently, MAP kinase phosphatase-1 (MKP-1) has been identified as a hypoxia responsive gene, but its role in the cardiomyocytes is poorly understood. MKP-1 is related to activation of mitogen-activated protein kinases (MAP kinases), and MAP kinases signaling pathways are important regulators of stress responsiveness. Heat shock protein (Hsp) functions to protect cells and to mediate an accelerated recovery following damage. Especially, it was known that Hsp70 and Hsp27 have the protective effects against hypoxic insult in cardiomyocytes. This study was to test the hypothesis of a possible crosstalk and the roles of MKP-1 and Hsp70 in hypoxia-induced cell death in cardiomyocytes. For these studies, using primary neonatal rat cardiomyocytes, we investigated the interconnectivity between hypoxia-induced cardiac cell death and MAP kinase signaling, MKP-1 and Hsp70 in vitro. The MAP kinases (ERK1/2, JNK and p38) level assessed by immunoblot analysis was inactivated at early time of hypoxia condition in cardiomyocytes. At this time MKP-1 and Hsp70 was accumulated. Pretreatment of cardiomyocytes with a MKP-1 inhibitor, vanadate, significantly recovered the ERK1/2 phosphorylation under the hypoxic condition. Interestingly, when cardiomyocytes was treated with Hsp70 inhibitor, MKP-1 expression level was significantly increased whereas ERK1/2 phosphorylation was reduced. To confirm these data, we used the recombinant Hsp70 protein fused with a protein transduction domain TAT. After Hsp70 transduction into cardiomyocytes, MKP-1 expression dramatically was reduced. These data suggest that MKP-1 expression is involved in hypoxia-induced cardiac cell death and this correlated precisely with ERK1/2 activity, and these regulations is associated with Hsp70.