Complement factor B (Bf) plays an important role in activating the alternative complement pathway. The inflammatory cytokines, in particular TNF-α and IFN-γ, are critical in the regulation of Bf gene expression in macrophages. In this study, we investigated the mechanisms of Bf gene regulation by TNF-α and IFN-γ in murine macrophages. Northern analysis revealed that Bf mRNA expression was synergistically up-regulated by TNF-α and IFN-γ in MH-S cells. Truncations of the 5′ Bf promoter identified a region between− 556 and− 282 bp that mediated TNF-α responsiveness as well as the synergistic effect of TNF-α and IFN-γ on Bf expression. Site-directed mutagenesis of a NF-κB-binding element in this region (− 433 to− 423 bp) abrogated TNF-α responsiveness and decreased the synergistic effect of TNF-α and IFN-γ on Bf expression. EMSAs revealed nuclear protein binding to this NF-κB cis-binding element on TNF-α stimulation. Supershift analysis revealed that both p50 and p65 proteins contribute to induction of Bf by TNF-α. An I-κB dominant negative mutant blocked Bf induction by TNF-α and reduced the synergistic induction by TNF-α and IFN-γ. In addition, the proteasome inhibitor MG132, which blocks NF-κB induction, blocked TNF-α-induced Bf promoter activity and the synergistic induction of Bf promoter activity by TNF-α and IFN-γ. LPS was found to induce Bf promoter activity through the same NF-κB cis-binding site. These findings suggest that a NF-κB cis-binding site between− 433 and− 423 bp is required for TNF-α responsiveness and for TNF-α-and IFN-γ-stimulated synergistic responsiveness of the Bf gene.