Previously, we designed and synthesized a new NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ). In the present research we looked into the effect of DHMEQ on the activation of macrophages, especially on the phagocytotic activity of cells of the mouse macrophage-like cell line RAW264.7. DHMEQ inhibited lipopolysaccharide (LPS)-induced NF-κB activation by inhibiting its nuclear translocation from the cytoplasm. It also inhibited the expression of inducible NO synthase (iNOS) and nitric oxide (NO) production induced by LPS and interferon-γ. Using enzyme-linked immunosorbent assays (ELISAs) we showed DHMEQ to inhibit LPS-induced secretion of IL-6, IL-12, interleukin-1β (IL-1β), and TNF-. Furthermore, DHMEQ also inhibited the phagocytosis of fluorescently labeled Escherichia coli by RAW264.7 cells treated with LPS or IL-1β, thus being the first evidence for the involvement of NF-κB in the regulation of phagocytosis by use of this inhibitor. Deletion of p65 by siRNA also inhibited the phagocytosis. DHMEQ inhibited the LPS-induced but not IL-1β-induced phagocytosis of glass beads, indicating that activation of not only NF-κB but also Toll-like receptor 4 (TLR-4) is essential for the phagocytosis of E. coli. Previously we found that DHMEQ inhibited type 2 collagen-induced rheumatoid arthritis and the growth of various human carcinomas in mice. It is thus likely that inhibition of macrophage activation is involved in the mechanism of these anti-inflammatory and antitumor activities of DHMEQ in mice.