Background: Ulinastatin (UTI) is a urinary trypsin inhibitor extracted and purified from urine of males. This study aimed to explore the effects of UTI on paraquat-induced-oxidative stress in human type II alveolar epithelial cells., Methods: The human type II alveolar epithelial cells, A549 cells, were cultured in vitro. The A549 cells were treated with different concentrations of paraquat (200, 400, 600, 800, 1 000, 1 200 µmol/L) and ulinastatin(0, 2 000, 4 000, 6 000, 8 000 U/mL) for 24 hours, the cell viability was measured by cell counting kit-8 and the median lethal concentration was selected. In order to establish an in vitro model of paraquat intoxication and to determine the safe dose of ulinastatin, we calculated LD50 using cell counting kit-8 to determine the survival rate of the cells. A549 cells were divided into normal control group, paraquat group and paraquat+ulinastatin group. The levels of malondialdehyde (MDA) and myeloperoxidase (MPO) were detected by biochemistry colorimetry, while the level of reactive oxygen spies (ROS) was detected by DCFH-DA assay., Results: The survival rate of A549 cells treated with different concentrations of paraquat decreased in a concentration-dependent manner. Whereas there was no decrease in the survival rate of cells treated with 0-4 000 U/mL ulinastatin. The levels of MDA, MPO, and ROS were significantly higher in the paraquat group than in the normal control group after 24-hour-exposure. And the survival rate of the paraquat+ulinastatin group was higher than that of the paraquat group, but lower than that of the normal control group. The levels of MDA, MPO, and ROS were lower than those of the paraquat group., Conclusion: Ulinastatin can alleviate the paraquat-induced A549 cell damage by reducing oxidative stress.