Previous studies have demonstrated the interaction between the Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) and the metastatic suppressor Nm23-H1 both in vitro and in vivo (C. Subramanian, M. A. Cotter II, and E. S. Robertson, Nat. Med. 7:350-355, 2001). Importantly EBNA3C can reverse the ability of Nm23-H1 to suppress migration of human cells in vitro. EBNA3C contributes to EBV-associated human cancers by regulating transcription of a number of cellular and viral promoters as well as targeting and altering the transcription activities of the metastasis suppressor Nm23-H1. Furthermore, Necdin is a cellular protein which is highly induced in terminally differentiated cells; it contributes to the regulation of cell growth and is also known to interact with viral oncoproteins. In this report, we show that Nm23-H1 and EBNA3C can modulate the biological functions of Necdin in the context of EBV infection and transformation. The levels of Necdin were consistently lower in EBV-positive cells, and EBNA3C could change the subcellular localization of Necdin as well as rescue cells from the antiangiogenic and antiproliferative effects mediated by Necdin. We also show that Necdin directly interacts with Nm23-H1, resulting in modulation of the biochemical function of Nm23-H1 as well as the biological function of Necdin. Both EBNA3C and Nm23-H1 were able to rescue not only Necdin-mediated transcriptional repression of the downstream vascular endothelial growth factor promoter but also Necdin-mediated growth suppression and antiangiogenic effects on cancer cells. The majority of this response was mediated through amino acid residues 191 to 222 of Necdin, which are also known to be important for nuclear matrix targeting. These studies suggest a role for Necdin in the regulation of downstream cellular targets in a hypoxic environment in virus-associated human cancers.