Endothelial nitric oxide synthase (eNOS) is a dynamic enzyme tightly controlled by co- and post-translational lipid modifications, phosphorylation and regulated by protein–protein interactions. In this study we have pharmacologically modulated the activation of eNOS, at different post-translational levels, to assess the role of eNOS-derived NO and regulatory mechanisms in tissue damage associated with spinal cord injury (SCI). SC trauma was induced by the application of vascular clips (force of 24 g) to the dura via a four-level T5–T8 laminectomy. SCI in mice resulted in severe trauma characterized by oedema, neutrophil infiltration, and production of inflammatory mediators, tissue damage and apoptosis. LY294002, an inhibitor of phosphatidylinositol 3-kinase that initiates Akt-catalysed phosphorylation of eNOS on Ser1179, was administered 1 h before the induction of SCI; 24 h after SCI sections were taken for histological examination and for biochemical studies. In this study we clearly demonstrated that pre-treatment with LY294002 reversed the increased activation of eNOS and Akt observed following SCI, and developed a severe trauma characterized by oedema, tissue damage and apoptosis (measured by TUNEL staining, Bax, Bcl-2 and Fas-L expression). Histological damage also correlated with neutrophil infiltration, assessed as myeloperoxidase activity. Overall these results suggest that activation of the Akt pathway in SC tissue subject to SCI is a protective event, triggered in order to protect the injured tissue through a fine tuning of the endothelial NO pathway.