The immature heart is known to be resistant to ischemia-reperfusion (IR) injury, however key proteins engaged in phospho-dependent signaling pathways crucial to cell survival are not yet defined. Our goal was to determine the post-natal changes in myocardial tolerance to IR, including baseline expression of key proteins governing IR tolerance and their phosphorylation during IR. Hearts from male C57Bl/6 mice (neonates, 2, 4, 8, and 12 weeks of age, n=6/group) were assayed for survival signaling/effectors (Akt, p38MAPK, GSK3β, HSP27, connexin-43, HIF1α, caveolin-3), regulators of apoptosis (Bax, Bcl-2) and autophagy (LC3B, Parkin, Beclin1). The effect of IR on ventricular function was measured in isolated perfused hearts from immature (4 week) and adult (12 week) mice. Neonatal myocardium exhibits a large pool of inactive Akt; high phospho-activation of p38MAPK, HSP27 and connexin-43; phospho-inhibition of GSK3β; and high expression of caveolin-3, HIF1α, LC3B, Beclin1, Bax and Bcl-2. Immature hearts sustained less dysfunction and infarction following IR than adults. Emergence of IR intolerance in adult vs. immature hearts was associated with complex proteomic changes: decreased expression of Akt, Bax and Bcl-2; increased GSK3β, connexin-43, HIF1α, LC3B and Bax:Bcl-2; enhanced post-ischemic HIF1α, caveolin-3, Bax and Bcl-2; and greater post-ischemic GSK3β and HSP27 phosphorylation. Neonatal myocardial stress-resistance reflects high expression of pro-survival and autophagy proteins, and apoptotic regulators. Notably, there is high phosphorylation of GSK3β, p38MAPK, HSP27, and low phosphorylation of Akt (high Akt 'reserve'). Subsequent maturational reductions in IR tolerance are associated with reductions in Akt, Bcl-2, LC3B and Beclin1.