We examined the effect of subchronic hypobaric hypoxia in rat heart. Adult male Sprague-Dawley rats were exposed at 25,000 ft for different time periods (2 and 5 days). Susceptibility of their hearts to oxidative stress as well as modulation in gene expression was evaluated. The results showed a crosstalk between reactive oxygen species (ROS) and nitric oxide (NO), initial response was accompanied by increase in ROS generation and development of oxidative stress as confirmed by increased lipid peroxidation, protein oxidation and accumulation of 2, 4-dinitrophenyl hydrazine and 4-hydroxy-2-nonenal adducts. At the same time, glutathione activity decreased; however, antioxidant enzymatic activities of superoxide dismutases, glutathione-S-transferase, and glutathione peroxidase rose in response to 5-days hypoxia. Interestingly, NO level increased till 5 days, however ROS decreased after 5 days; this observation suggests that ROS/NO balance plays an important role in cardioprotection. This observation is further supported by upregulation of antioxidant genes hemeoxygenase (HO-1) and metallothionein (MT). In addition, hypoxia also induces gradual upregulation of hypoxia-inducible transcription factor (HIF-1α), which in turn induces the expression of adaptive genes erythropoiesis, vascular endothelial growth factor, glucose transporter-1, nitric oxide synthase. Collectively, our data suggests a reciprocal regulation of ROS and NO and this effect is mediated by the increase in antioxidant proteins HO-1 and MT. Along with this HIF-1-mediated induction of various cardioprotective genes also plays an important role in acclimatization.