The protective mechanisms of hypoxic preconditioning (HPC) involve the mitigation of cellular calcium overload in cardiomyocytes. The sarcoplasmic reticulum (SR) chaperone calreticulin (CRT) plays an important role in regulating calcium homeostasis and is upregulated by HPC. The goal of this study was to show whether the late cardioprotection of HPC is mediated by calreticulin upregulation and to demonstrate whether the calreticulin induction is mediated by p38 MAPK phosphorylation. Hypoxic preconditioning was induced by hypoxemic hypoxic exposure by a 24-h period of normoxic reoxygenation before undergoing LAD occlusion in rats or hypoxia/reoxygenation (H/R) in cardiomyocytes. Ca uptake and release of the SR vesicles was determined by use of Ca and the Millipore filtration technique. Western blotting analysis was used to detect calreticulin expression and activity of p38 MAPK. Hypoxic preconditioning induced calreticulin upregulation and attenuated H/R injury in neonatal cardiomyocytes and myocardial ischemia injury by increasing calcium uptake and reducing calcium release in SR. Hearts from the HPC group were more resistant to sustained ischemia and had much stronger phosphorylation of p38 MAPK than sham operation. Inhibition of p38 MAPK with SB202190 (a selective p38 MAPK inhibitor) abolished the calreticulin upregulation and cardioprotection by HPC. Hypoxic preconditioning upregulates calreticulin expression through a p38 MAPK signaling pathway and protects cardiomyocytes from H/R (and ischemia) injury.