Mechanical traumatic injury causes cardiomyocyte apoptosis and cardiac dysfunction. However, the signaling mechanisms leading to post-traumatic cardiomyocyte apoptosis remains unclear. The present study attempted to identify the molecular mechanisms responsible for cardiomyocyte apoptosis induced by trauma. Normal cardiomyocytes (NC) or traumatic cardiomyocytes (TC, isolated immediately after trauma) were cultured with normal plasma (NP) or traumatic plasma (TP, isolated 1.5 hours after trauma) for 12 hours and apoptosis was determined by caspase-3 activation. Exposure of TC to NP failed to induce significant cardiomyocyte apoptosis. In contrast, exposure of NC to TP resulted in a greater than 2-fold increase in caspase-3 activation (P<0.01). Incubation of cardiomyocytes with cytomix (a mixture of TNF, IL-1 and IFN-) or TNF alone, but not with IL-1 or IFN-alone, caused significant caspase-3 activation (p<0.01). Trauma plasma-induced caspase-3 activation was virtually abolished by an anti-TNF antibody and trauma plasma isolated from TNF^-/- mice failed to induce caspase-3 activation. Moreover, incubation of cardiomyocytes with trauma plasma upregulated iNOS/NADPH oxidase expression, increased NO/superoxide production, and increased cardiomyocyte protein nitration (measured by nitrotyrosine content). These oxidative/nitrative stresses and resultant cardiomyocyte caspase-3 activation can be blocked by neutralization of TNF (anti-TNF antibody), inhibition of iNOS (1400W) or NADPH oxidase (apocynin), and scavenging of peroxynitrite (FP15) (P<0.01). Taken together, our study demonstrated that there exists a TNF-initiated, cardiomyocyte iNOS/NADPH oxidase-dependent, peroxynitrite-mediated signaling pathway that contributes to post-traumatic myocardial apoptosis. Therapeutic interventions that block this signaling cascade may attenuate post-traumatic cardiac injury and reduce the incidence of secondary organ dysfunction after trauma.