Latent membrane protein‑1 (LMP-1) of Epstein‑Barr virus (EBV) promotes tumorigenesis. Here, we report that LMP-1 activates the immunoregulatory molecule CD69 gene transcription through a nuclear factor-κB (NF-κB)‑dependent pathway. CD69 expression was upregulated in LMP-1‑expressing EBV-immortalized human B-cell lines and an EBV-positive Burkitt's lymphoma cell line. LMP-1 expression increased CD69 expression at the transcriptional level. CD69 promoter was regulated by LMP-1 activation of NF-κB via the carboxy-terminal activation region 1 and 2. Promoter deletion analysis indicated that two NF-κB binding sites are necessary for activation of the CD69 promoter. Electrophoretic mobility shift analysis demonstrated that LMP-1 activates both NF-κB binding sites in the CD69 promoter. This is the first report of the regulation of CD69 expression by LMP-1, and this novel finding may, thus, represent an important link between the EBV oncoprotein LMP-1 and its critical role in the development of EBV-associated diseases.