Artemisia vestita Wall., a traditional Tibetan medicine, has wide clinical application for inflammatory diseases. However, its molecular mechanism of anti-inflammatory effect is poorly understood. In the present study, we investigated the anti-inflammatory activity and under-lying mechanism of the ethanol extract from Artemisia vestita (AV-ext) on lipopolysaccharide (LPS)-induced sepsis. Pretreatment with AV-ext significantly decreased the levels of tumor necrosis factor-α (TNF-α) in serum and liver and lung tissues, and improved the survival of mice with experimental sepsis. AV-ext also remarkably reduced the expression levels of TNF-α, interleukin-1β and cyclooxygenase-2 in LPS-stimulated RAW 264.7 macrophages and dose dependently suppressed the activation of mitogen-activated protein kinases (MAPKs), such as p38, extracellular signal-regulated kinase (ERK1/2) and c-Jun NH2-terminal kinase (JNK). Furthermore, pretreatment with AV-ext dose dependently inhibited the activation of nuclear factor-κB (NF-κB), as well as the degradation and phosphorylation of inhibitory κB (IκB) in LPS-activated RAW 264.7 macrophages. These results collectively reveal that AV-ext inhibits TNF-α release from macrophages by suppressing MAPK and NF-κB signaling pathways and suggest that AV-ext may be beneficial for the treatment of endotoxin shock or sepsis.