Objective To investigate the safety and efficacy of human umbilical cord mesenchymal stem cells (HUC-MSCs) in treating lipopolysaccharide (LPS) induced acute lung injury in a mouse model. Methods The safety of HUC-MSCs was evaluated through tumorigenicity experiments, in vitro hemolysis experiments, and acute toxicity experiments. A model of acute lung injury was constructed by nasal or intraperitoneal injection of LPS in mice aged 6 to 8 weeks. HUC-MSCs treatment was given 6 h after modeling, and the treatment groups were divided into tail vein injection group (5×10⁷ cells/ kg) and nebulized group (HUC-MSCs conditioned medium). The histopathological result of mice lungs in each group were observed 96 h after modeling, and the inflammatory cell count and classification of bronchoalveolar lavage fluid ( BALF ) were observed by Richter-Kimsa staining. Results There was the absence of potentially tumorigenicity and acute toxicity in the animals after administration of the stem cells.HUC-MSCs had no hemolytic effect on rabbit blood. Compared with the control group, both modeling method showed some degree of lung injury, and the McGuigan score of lung histopathological sections showed that the lung injury caused by intraperitoneal injection of LPS was more severe than that of the nasal drip group. Compared with the model group, the degree of lung injury was significantly reduced in the treatment group, in which the tail vein injection group had a better treatment effect than the atomization group, and the macrophage count in BALF was significantly higher (P<0.001). Conclusions The mouse model of acute lung injury induced by LPS intraperitoneal injection was superior to nasal drip administration. Caudal vein injection of HUC- MSCs can effectively treat acute lung injury in mice. HUC-MSCs conditioned medium atomization can alleviate lung inflammation in mice.