Transmembrane TNF-alpha (tmTNF-alpha) and secretory TNF-alpha (sTNF-alpha) display opposite effects in septic shock. Reducing tmTNF-alpha shedding can offset the detrimental effects of sTNF-alpha and increase the beneficial effect of tmTNF-alpha. We previously developed a monoclonal antibody that is specific for tmTNF-alpha and does not cross-react with sTNF-alpha. In this study, we show that this antibody can specifically suppress tmTNF-alpha shedding by competing with a TNF-alpha converting enzyme that cleaves the tmTNF-alpha ectodomain to release sTNF-alpha This tmTNF-alpha antibody significantly inhibited LPS-induced secretion of interleukin (IL)-1 beta, IL-6, interferon-beta, and nitric oxide by monocytes/macrophages, and protected mice from septic shock induced by lipopolysaccharide (LPS) or cecal ligation and puncture, while reducing the bacterial load. The mechanism associated with the protective effect of this tmTNF-alpha antibody involved promotion of LPS-induced toll-like receptor 4 (TLR4) internalization and degradation by recruiting Triad3A to TLR4. Moreover, the tmTNF-alpha antibody inhibited LPS- induced activation of nuclear factor-kappa B and interferon regulatory factor 3 pathways by upregulating expression of A20 and monocyte chemotactic protein-induced protein 1. Similarly, treatment of macrophages with exogenous tmTNF-alpha suppressed LPS/TLR4 signaling and release of proinflammatory cytokines, indicating that increased levels of tmTNF-alpha promoted by the antibody contributed to its inhibitory effect. Thus, use of this tmTNF-alpha antibody for specific suppression of tmTNF-alpha shedding may be a promising strategy to treat septic shock.