Equine endotoxemia is a serious clinical problem with high mortality. Only a few treatments have been proved the therapeutic efficacy for equine endotoxemia. Excessive nuclear factor-kappa B (NF-κB) activation and production of proinflammatory cytokines, which were induced by reaction to lipopolysaccharide (LPS), would play a key role in the pathogenesis of endotoxemia. Bortezomib, a proteasome inhibitor, inhibits NF-κB signaling pathway through blocking proteasomal degradation of NF-κB inhibitor alpha. This study aimed to evaluate the effect of bortezomib on tumor necrosis factor-alpha (TNF-α) production by LPS-stimulated equine monocytes in vitro and on clinical and inflammatory parameters in an in vivo endotoxemia model. Bortezomib significantly inhibited LPS-induced TNF-α production through inhibition of NF-κB activity in vitro. In a cross over design, horses received pretreatment of either bortezomib (1.3 mg/m2) or vehicle (dimethyl sulfoxide) before the infusion of 30 ng/kg LPS. Clinical parameters including behavioral pain scores and hoof wall surface temperature (HWST) were measured over 7 hours. In an endotoxemia model, bortezomib had a tendency to improve painful reaction and reduction of HWST. Bortezomib would have a potential as a therapeutic agent for equine endotoxemia.