Studies have shown that p38 MAPK and nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) play key roles under physiological and pathophysiological conditions. Although administration of 17-estradiol (E2) protects cardiovascular injury from trauma-hemorrhage, the mechanism by which E2 produces those effects remains unknown. Our objective was to determine whether the E2-mediated activation of myocardial p38 MAPK and subsequent eNOS expression/phosphorylation would protect the heart following trauma-hemorrhage. To study this, male Sprague-Dawley rats underwent soft-tissue trauma (midline laparatomy) and hemorrhagic shock (mean blood pressure 35-40 mmHg for 90 min) followed by fluid resuscitation. Animals were pretreated with specific p38 MAPK inhibitor SB203580 (SB, 2 mg/kg), and nonselective NOS inhibitor L-NAME (30 mg/kg) 30 min before vehicle (cyclodextrin) or E2 (100 µg/kg) treatment followed by resuscitation and sacrificed 2 hrs thereafter. Cardiovascular performance and other parameters were measured. E2 administration following trauma-hemorrhage increased cardiac p38 MAPK activity, eNOS expression/phosphorylation at serine ¹¹⁷⁷, and increased nitrate/nitrite levels in plasma and heart tissues; these were associated with normalized cardiac performance, which was reversed by SB administration. In addition, E2 also prevented trauma-hemorrhage-induced increase in cytokines (IL-6 and TNF-), chemokines (MIP-2 and CINC-1) and ICAM-1, which was reversed by L-NAME administration. Administration of E2 following trauma-hemorrhage attenuated cardiac tissue injury markers, myeloperoxidase activity and nitrotyrosine level, which were reversed by treatment with SB and L-NAME. The salutary effects of E2 on cardiac functions and tissue protection following trauma-hemorrhage are mediated in part through activation of p38 MAPK and subsequent eNOS expression/phosphorylation.