Although anoxic preconditioning (APC) in the myocardium has been investigated for many years, its physiological mechanism is still not completely understood. Increasing evidence indicates that transiently increased resistance to ischemic damage following APC is dependent on de novo proteins synthesis. However, the key effector pathway(s) associated with APC still remains unclear. The proto-oncogene Pim kinase belongs to a serine/threoine protein kinase family, consists of Pim-1, Pim-2 and Pim-3 and has been implicated in stimulating cell growth and inhibiting cell apoptosis. Therefore we assumed that Pim-3 expression might be aberrantly induced in cardiomyocytes that were subjected to anoxia/reoxygenation (A/R) injury and that Pim-3 might also contribute to cardio-protection after APC. To address this hypothesis, we cloned a Pim-3 expression vector, transfected it into rat cardiomyocytes, and examined Pim-3 expression in rat cardiomyocytes that were subjected to A/R injury. Moreover, we studied the role of three major MAPK pathways, e.g. p38 MAPK, JNK, and ERK1/2, in order to evaluate the molecular mechanism underlying Pim-3 up-regulation and A/R induced cardiomyocyte injury. Our experiments showed that APC induced an up-regulation of Pim-3 and the transfection of Pim-3 gene into the cardiomyocytes attenuated A/R injury. The inhibition of p38 MAPK by SB203580 abolished both the Pim-3 up-regulation and the cardio-protection provided by APC. Overall, these results suggest that APC could act to protect the heart from A/R injury with cooperation from the proto-oncogene Pim-3; in addition, it up-regulates Pim-3 expression through a p38 MAPK signaling pathway.