Defensins have a broad range of antimicrobial activity against bacteria, fungi, and viruses. The expression of human beta-defensin-2 (hBD-2) is prevalently observed in epithelial cells and is induced by bacterial infection. Here, we have shown that the expression of the hBD-2 gene and release of hBD-2 protein into the medium is up-regulated in response to CpG-DNA in human B cell line RPMI 8226. The induction of hBD-2 was dependent on CG sequence and phosphorothioate backbone-modification. This was also confirmed in primary human lymphocytes. To shed light on the molecular mechanism involved in hBD-2 induction by CpG-DNA, we examined the contribution of the NF-kappaB signaling pathway in RPMI 8226 cells. Suppression of MyD88 function and inhibition of NF-kappaB nuclear localization blocked hBD-2 induction. The NF-kappaB pathway inhibitors also abolished hBD-2 induction. These results may contribute to a better understanding on the therapeutic effects of CpG-DNA against infectious diseases.