As COVID-19 spreads over the world, the treatment of acute lung injury (ALI) has attracted much attention. Considering ubiquitin-specific protease (USP) 25 has been relevant to inflammation, this article focused on its role in ALI and its regulatory mechanism. Lipopolysaccharide (LPS) was applied to separately stimulate mice and human lung epithelial cells to establish in vivo and in vitro ALI models. To discover the effects of USP25 overexpression on mouse, lung pathology, inflammatory factor levels, edema, number of inflammatory cells, and downstream protein levels were evaluated. USP25 overexpression in mice could alleviate LPS-induced lung tissue lesions and edema, and reduce inflammatory factors and inflammatory cells. It also inhibited the levels of downstream TRAF6, MAPK pathway-related proteins, and Fos Proto-Oncogene (FOS) in vivo. Furthermore, BEAS-2B cells were transfected with TNF receptor-associated factor 6 (TRAF6) plasmids to study the role of TRAF6 in the regulatory mechanism of USP25. TRAF6 overexpression was found to reverse the functions of USP25 overexpression on cells. In conclusion, USP25 reduced ALI and inhibited inflammation in lung epithelial cells via regulating TRAF6/MAPK/FOS signaling.