Background Urinary trypsin Inhibitor inhibits the enhanced production of pro-inflammatory molecules. Hemeoxygenase-1 induction protects against ischemia/reperfusion injury, oxidative stress, inflammation, transplant rejection, apoptosis, and other conditions. However, it is unknown if a combined hemin and ulinastatin pretreatment could result in protective effects for septic shock. In this study, we investigated the role of hemin pretreatment combined with ulinastatin on septic shock in rats.Methods Eighty healthy, male Sprague-Dawley rats were randomly divided into four groups: group S, group H, group U and group HU. Groups S and U received 1 ml normal saline intraperitoneally, while groups H and HU both received 1 ml (100 mg /kg) hemin. Twenty-four hours later, 0.5 ml (10 mg/kg) E. Coli lipopolysaccharide was injected intravenously to replicate the experimental model of septic shock. After an initial 25% decrease in the mean arterial pressure, corresponding to time point 0, groups HU and U received 0.5 ml 10 000 U/kg ulinastatin intravenously, and the others received 0.5 ml normal saline.Results The number of deaths in groups H and U was Iower than that in the group S (P＜0.05), and was higher than that in group HU (all P＜0.05) respectively. The mean arterial pressure (MAP) in the group S was significantly greater than that in group H (P＜0.05), and was Iower than that in group HU and group U (P＜0.05). The plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr) and blood urea nitrogen (BUN), the malondial-dehyde (MDA) of liver, kidney and lung, and the lung Evans blue (EB) Contents in groups H and U, were greater than that in group HU (all P＜0.05), and were Iower than that in group S (all P＜0.05). In contrast, the plasma levels of CO in groups H and HU were higher than that in groups S and U (all P＜0.05), and SOD of liver, kidney and lung in groups H and U were higher than that in group S, and were Iower than that in group HU (all P＜0.05). The levels of TNF-α, IL-6, IL-8 and β-glucuronidase (GCD) activity of plasma in groups U and HU were Iower than those in groups H and S, all having a P＜0.05, while there were no significant differences between group H and group S, or between group HU and group U (all P＞0.05). The HO-1 mRNA and HO-1 protein levels from hepatic, renal, and pulmonary tissue in groups S and U were Iower than those in groups H and HU (all P＜0.05), but there were no significant differences between groups S and U, or between groups H and HU (all P＞0.05). The HO-2 mRNA and HO-2 protein were not significantly different among the four groups (all P＞0.05).Conclusions Combined pretreatment with hemin and ulinastatin in septic shock rats results in an improved response by the upregulation of HO-1 protein followed by increasing CO with resistance to increased oxidative stress, restraining the release of inflammatory mediators, and inhibiting β-GCD activity.