Macrophages are antigen-presenting cells (APCs) that play a crucial role in the innate immune response and may be involved in both clearance and spread of viruses. Stimulation of macrophages via Toll-like receptors (TLRs) results in activation of nuclear factor κB (NF-κB) and synthesis of proinflammatory cytokines. In this work, we show modulation of proinflammatory NF-κB signaling by a member of the family Poxviridae, genus Orthopoxvirus--ectromelia virus (ECTV)--in RAW 264.7 murine macrophages. ECTV interfered with p65 NF-κB nuclear translocation induced by TLR ligands such as lipopolysaccharide (LPS) (TLR4), polyinosinic-polycytidylic acid (poly(I:C)) (TLR3) and diacylated lipopeptide Pam2CSK4 (TLR2/6). We observed that ECTV modulates phosphorylation of Ser32 of inhibitor of κB (IκBα) and Ser536 of p65. Interference of ECTV with TLR signaling pathways implied that proinflammatory cytokine synthesis was inhibited. Our studies provide new insights into the strategies of proinflammatory signaling modulation by orthopoxviruses during their replication cycle in immune cells. Understanding important immune interactions between viral pathogens and APCs might contribute to the identification of drug targets and the development of vaccines.