Florfenicol, an antibiotic used to treat infection, has previously been shown to modulate early cytokine responses and increase mouse survival in endotoxemia. In the present study, we investigated in vivo the effect of florfenicol on acute lung injury (ALI) induced by lipopolysaccharide (LPS). In the mouse model of LPS-induced inflammatory lung injury, we found that pretreatment with a single 100mg/kg dose of florfenicol significantly decreases the W/D ratio of lungs and protein concentration in the bronchoalveolar lavage fluid (BALF) and significantly reduces the number of total cells, neutrophils and macrophages in the BALF at 24h after LPS challenge. In addition, histopathological examination indicates that florfenicol significantly attenuates tissue injury of the lungs in LPS-induced ALI. Furthermore, florfenicol also inhibits the production of several inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha) at 6 and 12h, interleukin-6 (IL-6) at 12 and 24h, and interleukin-1ss (IL-1ss) at 12h, in the BALF after LPS challenge. These results suggest that florfenicol protects against LPS-induced ALI in mice.