Although the murine late pregnant (LP) heart is speculated to be a better functioning heart during physiological conditions, the susceptibility of LP hearts to I/R injury is still unknown. The aims of this study were to investigate the cardiac vulnerability of LP rodents to ischemia/reperfusion (I/R) injury and to explore its underlying mechanisms. In vivo female rat hearts [non-pregnant (NP) or LP] or ex vivo Langendorff-perfused mouse hearts were subjected to I/R. The infarct size was approximately fourfold larger in LP animals compared with NP both in vivo and ex vivo. The heart functional recovery was extremely poor in LP mice compared with NP (~10% recovery in LP vs. 80% recovery in NP at the end of reperfusion, P < 0.01). Interestingly, the poor functional recovery and the larger infarct size in LP were partially restored one day post-partum and almost fully restored 1 week post-partum to their corresponding NP levels. Mitochondrial respiratory function and the threshold for opening of the mitochondrial permeability transition pore were significantly lower in LP compared with NP when they both were subjected to myocardial I/R injury [Respiratory control ratio = 1.9 ± 0.1 vs. 4.0 ± 0.5 in NP, P < 0.05; calcium retention capacity (CRC) = 167 ± 10 vs. 233 ± 18 nmol/mg protein in NP, P < 0.01]. Cardiac reactive oxygen species (ROS) generation, as well mitochondrial superoxide production, was approximately twofold higher in LP compared with NP following I/R. The phosphorylation levels of Akt, ERK1/2, and STAT3, but not GSK3β, were significantly reduced in the hearts from LP subjected to I/R. In conclusion, increased mitochondrial ROS generation, decreased CRC as well as impaired activation of Akt/ERK/STAT3 at reperfusion are the possible underlying mechanisms for higher vulnerability of LP hearts to I/R.