Ischemia-induced injury contributes to the progression of coronary artery disease. To date it is not known what governs ischemia-mediated perturbation of signaling in heart, or what strategy might be used to counteract such perturbation thus protecting heart against ischemic insults. It has been proposed that dysfunctions of protien kinases or phosphatases might lead to injury in heart suffering from ischemia. However, it remains elusive how ischemia-induced perturbations of intrinsic phosphorylation signaling contribute to cardiac damage. In the present study, using surgical ligation of the left anterior descending coronary artery (LAD) as a model, we have shown for the first time that the levels of phosphotyrosine (pTyr) signaling were decreased significantly in the left ventricular tissue of mouce heart, concomitant with a burst of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) released to the circulation. To gain insights into this disaese process at the molecular level, we examined signaling events in rat myocardial H9c2 cells. Interestingly, H9c2 cells underwent a drastic decrease of pTyr signaling in response to hypoxia. Such results recapitulated the ischemia-induced perturbation of signaling in heart. This change was associated with the activation of protein tyrosine phosphatases (PTPs) and a loss of cytoskeletal integrity. Employing a pan-PTP inhibitor phenyl vinyl sulfone in H9c2 cells, we showed a critical role of PTP activation in hypoxia-induced adverse effects. Furthermore, we identified that the hypoxic condition drives reduction of the active-site Cys residue of PTPs, leading to a significant increase of their activity. These observations led to a hypothesis that the intervention by nitric oxide (NO) might prevent PTP activation via S-nitrosylation on the active-site Cys residue, thus suppressing the hypoxic injury. Importantly, treatment with S-nitrosoglutathione (GSNO) in H9c2 cells not only sustained pTyr levels but also reversed hypoxia-induced cytoskeletal damages. We further showed that four PTPs, which control the function of cytoskeletal regulators, were indeed S-nitrosylated and inactivated by the GSNO treatment under the hypoxic condition. The application of GSNO therapy was tested in mice with LAD ligation. Intriguingly, ischemia-induced cardiac injury as evidenced by release of CPK and LDH was markedly attenuated by GSNO treatment, along with a significant rebound of pTyr signaling. Taken together, our data suggest a protective role of NO via inactivation of PTPs, thus providing a novel therapeutic opportunity for heart suffering from ischemic insult.