Oncological treatment is often accompanied by irradiation therapy, which even in the safe and well-established protocols has numerous adverse effects that require investigation in experimental models. Therefore, the aim of our study was to explore the effects of a single dosage of irradiation (25 Gy) applied on the chest area of normal adult Wistar rats. Our research was focused on evaluation functional parameters and changes of selected molecular markers 6 weeks after irradiation. Irradiation led to general alteration of the animals, e.g., body and heart weight retardation and presence of exudate in the chest cavity. In isolated Langendorff-perfused hearts, the effect of irradiation on heart function was manifested only by mild bradycardia and enhanced coronary flow, but no changes in heart contractile parameters were observed. Under conditions of ischemia/reperfusion (I/R), although the incidence of reperfusion arrhythmias was higher than in the control (C) non-irradiated hearts, recovery of functional parameters (LVDP) was not worsened as compared with C, moreover, the size of infarction in the irradiated hearts was even smaller than in the intact hearts. In addition, gene expression of PPARα (real-time RT-PCR) was significantly lower in left ventrucular tissue of irradiated rats, while expression of microRNA-21 in these hearts was increased nearly 10-fold. These findings suggest that cardioprotective molecular mechanisms that account for infarct size reduction could be activated in the irradiated animals. In conclusion, our results demonstrate that single dosage of irradiation may cause both negative and adaptative changes in the heart that make it more resistant to I/R.