Cot is a MAPK kinase kinase that has been implicated in cellular activation and proliferation. Here, we show that the addition of lipopolysaccharide (LPS) to RAW264 macrophages induces a 10-fold increase of endogenous Cot activity, measured as MAPK kinase kinase 1 activity. Taxol, but not phorbol 12-myristate 13-acetate (PMA), induces a similar activation of Cot. A tyrosine kinase activity is involved in Cot activation by LPS. 15-DeoxyΔ12,14-prostaglandin J2, but not rosiglitazone, blocks Cot activation by LPS. Furthermore, 15-deoxy-Δ 12,14-prostaglandin J2 also inhibited the LPS-induced Cot in vitro. However, 15-deoxy-Δ12,14-prostaglandin J2 does not inhibit MAPK kinase 1 or ERK1/ERK2 activation/phosphorylation induced by PMA and mediated by c-Raf. Considering these data, we propose that the inhibition of LPS-induced Cot activation is one mechanism by which 15-deoxy-Δ12,14-prostaglandin J2 acts as an anti-inflammatory.