Cytokines stimulate inflammatory defenses against viral infections. In order to evade host defenses, viruses have developed strategies to counteract antiviral cytokines. African swine fever virus (ASFV) is a large, double-stranded DNA virus that infects macrophages. This study demonstrates that ASFV effectively inhibited phorbol myristic acid-induced synthesis of antiviral, proinflammatory cytokines alpha interferon, tumor necrosis factor alpha, and interleukin-8 in infected macrophages as assessed by enzyme-linked immunosorbent assay and reverse transcriptase PCR. In contrast, levels of mRNA and protein for transforming growth factor beta, an anti-inflammatory cytokine, were increased by ASFV infection, suggesting that ASFV-induced inhibition of cytokine synthesis may be limited to cytokines activated by NFkappaB. An interleukin-8 promoter, containing an NFkappaB enhancer site, driving expression of a luciferase reporter gene was used to show that NFkappaB-dependent transcription was inhibited by the virus and by a cloned ASFV gene, A238L. This gene encodes a protein with homology to IkappaB, the inhibitor of NFkappaB. Electrophoretic mobility shift assay showed that cells expressing the A238L gene inhibited NFkappaB binding to DNA. These results suggest that the A238L gene product interacts with NFkappaB to prevent transcription and downregulate proinflammatory cytokine production. This novel viral evasion strategy encoded in a single IkappaB-like protein may be capable of inhibiting most macrophage NFkappaB-dependent antiviral mechanisms and may provide insights into how ASFV causes a fatal hemorrhagic disease of domestic pigs and a persistent infection in the African warthog, which is its natural permissive host.