ABSTRACT: Objectives: Hemorrhagic shock (HS) can initiate an exaggerated systemic inflammatory response and multiple organ failure, especially if followed by a subsequent inflammatory insult (“second hit”). We have recently shown that histone deacetylase inhibitors can improve survival in rodent models of HS or septic shock, individually. In the present study, we examined whether valproic acid (VPA), a histone deacetylase inhibitor, could prolong survival in a rodent “two-hit” model: HS followed by septic shock from cecal ligation and puncture (CLP). Methods: Male Sprague-Dawley rats (250–300 g) were subjected to sublethal HS (40% blood loss) and then randomly divided into two groups (n = 7/group): VPA and control. The VPA group was treated intraperitoneally with VPA (300 mg/kg in normal saline [NS], volume = 750 μL/kg). The control group was injected with 750 μL/kg NS. After 24 h, all rats received CLP followed immediately by injection of the same dose of VPA (VPA group) or NS (vehicle group). Survival was monitored for 10 days. In a parallel study, serum and peritoneal irrigation fluid from VPA- or vehicle-treated rats were collected 3, 6, and 24 h after CLP, and enzyme-linked immunosorbent assay was performed to analyze myeloperoxidase activity and determine tumor necrosis factor α and interleukin 6 concentrations. Hematoxylin-eosin staining of lungs at 24-h time point was performed to investigate the grade of acute lung injury. Results: Rats treated with VPA (300 mg/kg) showed significantly higher survival rates (85.7%) compared with the control (14.3%). Moreover, VPA significantly suppressed myeloperoxidase activity (marker of neutrophil-mediated oxidative damage) and inhibited levels of proinflammatory cytokine tumor necrosis factor α and interleukin 6 in the serum and peritoneal cavity. Meanwhile, the severity of acute lung injury was significantly reduced in VPA-treated animals. Conclusions: We have demonstrated that VPA treatment improves survival and attenuates inflammation in a rodent two-hit model.