beta-Defensins are cationic peptides with broad-spectrum antimicrobial activities that contribute to innate host defense. Among human beta-defensins (hBDs), hBD-2 is up-regulated in epithelial tissues and mononuclear phagocytes in response to bacterial infection and proinflammatory cytokines. However, little is known about the molecular mechanism of hBD-2 gene regulation. Here, we investigated lipopolysaccharide (LPS)-mediated transcriptional regulation of the hBD-2 gene by focusing on the roles of NF-kappa B, STAT, and NF-IL-6 sites in mononuclear phagocytes using RAW264.7 cells, which are sensitive to LPS. Luciferase reporter analyses demonstrated that two NF-kappa B sites were essential for full LPS responsiveness of the hBD-2 gene. Further, both NF-kappa B sites were also crucial for basal transcriptional activity. In contrast, neither the NF-IL-6 nor STAT binding site was required for LPS-induced hBD-2 transcription. Electrophoretic mobility shift assay indicated that in unstimulated cells, NF-kappa B p50 homodimer bound to both NF-kappa B sites, whereas the p65-p50 heterodimer formed complexes with these sites following LPS stimulation. Together, these observations indicate that NF-kappa B plays an important role in the regulation of hBD-2 gene expression in response to LPS.