Chemically modified tetracyclines are orally active inhibitors of multiple proteases and cytokines. In this study, we focused on the regulation of tumor necrosis factor (TNF)-alpha and mitogen-activated protein kinases (MAPKs) in sepsis and their reduction by treatment with nonantimicrobial chemically modified tetracycline-3 (CMT-3), which retains their antiinflammatory activity. Sepsis was induced in rats by cecal ligation and puncture (CLP). At 24 h and 1 h before CLP, treated rats received CMT-3 (25 mg/kg), and untreated rats received saline by gavage. At 0 h, 0.5 h, 1.5 h, and 24 h after CLP, blood and liver samples were collected. TNF-alpha was determined by ELISA, and MAPKs were determined by Western blot analysis. A significant activation of p38 MAPK was observed after 0.5 h and 1.5 h of sepsis that appeared to coincide with the increased circulating TNF-alpha level. The activation of p42/44 was increased after 24 h of sepsis, whereas that of SAPK/JNK was unaltered throughout the course of sepsis. CMT-3 pretreatment inhibited the TNF-alpha level as well as p38 MAPK activation seen after 0.5 and 1.5 h of CLP and also suppressed the activation of p42/44 after 24 h post-CLP. These results indicate increased activity of TNF-alpha and MAPK following sepsis and demonstrate the beneficial effect of CMT-3 in preventing the increase in TNF-alpha, p38 MAPK, p42/44 MAPK, and the progression of septic shock.