The aim of this study is to research and compare levels of MMP-9, TNF-alpha, IL-6, IL-8 and IL-10 through different doses of ulinastatin ( UTI). Furthermore, the mechanism of UTI against acute lung injury (ALI) induced by sepsis was explored using methods of cecal ligation and puncture. An animal model was established, in which 78 rats were randomly divided into three groups (n = 26): CLP group (ALI group), UTI 50,000 U/kg group (UTI1 group), and UTI 100,000 U/kg group (UTI2 group); six rats were treated as controls. A corresponding dose of UTI was given immediately after surgery in the UTI treatment groups. Blood samples and lung tissues were collected. Survival rate, lung wet weight index, pathology scores of the lung, as well as MMP-9, TNF-alpha, IL-6, IL-8 and IL-10 serum levels were detected at every time point. MMP-9, TNF-alpha, IL-8, IL-6 and IL-10 were determined by double antibody sandwich ELISA assay, and statistical analysis was performed. After using UTI intervention, lung injury was alleviated and no significant change in survival rate occurred. Furthermore, inflammatory mediators declined in each group, in which both the UTI1 and UTI2 groups had a descending trend compared with the ALI group; and this significantly decreased compared with the 48 h ALI group (P < 0.01). TNF-alpha markedly increased in the ALI group, but this trend decreased in the UTI1 and UTI2 groups. IL-8 was elevated at every point in the ALI group, but this significantly decreased in the UTI1 and UTI2 groups, compared with the 48 h ALI group (P < 0.01). The UTI1 and UTI2 groups revealed a descending trend compared with the ALI group, and the UTI2 group significantly decreased compared with the 24 h ALI group ( P < 0.05). Both the UTI1 and UTI2 groups deceased compared with the ALI group, although there was no significant difference. The most significant decrease was observed in MMP- 9 and IL-8 ( P < 0.01). UTI plays a protective role against lung injury by inhibiting inflammatory factors such as MMP-9 and IL-8. This study may provide a new clue in the treatment of ALI vertical bar.