Background: Neonatal hearts are less susceptible to developing myocardial dysfunction after hypoxia and/or ischemia than adult hearts. Differences in intracellular calcium homeostasis may be responsible for reduced calcium overload of the immature myocardium leading to the observed protection against ischemia. Objective: To assess differences in baseline and post-ischemic gene expression of calcium handling proteins after ischemia in neonatal and adult rabbit hearts. Methods: We used isolated antegrade perfused rabbit hearts (age 2 days, 28 days, n = 32), which were exposed to ischemia and hypothermia simulating myocardial stunning comparable to neonatal asphyxia. Gene and protein expression of the sodium–calcium exchanger (NCX), the sarco-endoplasmatic reticulum Ca2+-ATPase 2a (SERCA) and calsequestrin (CSQ) were measured using quantitative real-time PCR and Western blotting. Results: After ischemia and reperfusion in neonatal and adult hearts, a significant decrease in myocardial performance was recorded. At the mRNA level, significant differences in the baseline expression of NCX, SERCA and CSQ between neonatal and adult hearts were observed. In neonatal post-ischemic hearts, NCX and CSQ expression were significantly higher at the mRNA level than in controls. In contrast, SERCA expression remained unchanged in neonatal hearts and decreased in adult hearts compared to the non-ischemic controls. Conclusion: These findings suggest that changes in gene expression of calcium handling proteins may be involved in the different susceptibility of neonatal compared to adult hearts to developing myocardial dysfunction after ischemia.