Oxidant injury contributes to acute lung injury (ALI). We previously reported that activation of protein kinase GI (PKG_I) post-transcriptionally increased the key antioxidant enzymes catalase and glutathione peroxidase 1 (Gpx-1) and attenuated oxidant-induced cytotoxicity in mouse lung microvascular endothelial cells (MLMVEC). The present studies tested the hypothesis that the antioxidant effect of PKG_I is mediated via inhibition of the c-Abl tyrosine kinase. We found that activation of PKG_I with the cGMP analogue 8pCPT-cGMP inhibited c-Abl activity and decreased c-Abl expression in wild type but not PKG_I-/- MLMVEC. Treatment of wild-type MLMVEC with atrial natriuretic peptide also inhibited c-Abl activation. Moreover, treatment of MLMVEC with the c-Abl inhibitor imatinib increased catalase and GPx-1 protein in a post-transcriptional fashion. In imatinib-treated MLMVEC, there was no additional effect of 8pCPT-cGMP on catalase or GPx-1. The imatinib-induced increase in antioxidant proteins was associated with an increase in extracellular H2O2 scavenging by MLMVEC, attenuation of oxidant-induced endothelial barrier dysfunction, and prevention of oxidant-induced endothelial cell death. Finally, in the isolated perfused lung, imatinib prevented oxidant-induced endothelial toxicity. We conclude that cGMP, through activation of PKG_I, inhibits c-Abl, leading to increased key antioxidant enzymes and resistance to lung endothelial oxidant injury. Inhibition of c-Abl by active PKG_I may be the downstream mechanism underlying PKG_I-mediated antioxidant signaling. Tyrosine kinase inhibitors may represent a novel therapeutic approach in oxidant-induced ALI.