Some antibacterials have been reported to regulate the host immune and inflammatory responses both in vitro and in vivo. Florfenicol is an antibiotics used in treatment of infection. We investigated the effects of florfenicol on cytokine production by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages in vitro, and the results showed that florfenicol reduced tumor necrosis factor (TNF) and interleukin-6 (IL-6) production but had little effect on interleukin-1beta (IL-1beta) and interleukin IL-10 (IL-10) secretion. This inspired us to further study the effects of florfenicol in vivo. Florfenicol significantly attenuated TNF and IL-6 production in serum from mice challenged with LPS, and in consistent with the results in vitro. In murine model of endotoxemia, mice were prophylactically or therapeutically treated with florfenicol prior to or after LPS challenge. The results showed that florfenicol significantly increased mouse survival. Further studies revealed that florfenicol prevented the LPS-induced nuclear factor-kappaB (NF-kappaB) translocation from cytoplasm into nuclear in RAW 264.7 macrophages. These observations indicate that florfenicol modulates early cytokine responses by blocking NF-kappaB pathway, and thus, increases mouse survival. This effect of the drug may be of potential usefulness in treatment of bacterial shock.