Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin homology (PH) and Src homology 2 (SH2)-like domains, as well as a proline-rich domain in its C-terminal region. In previous studies, we demonstrated that STAP-2 binds to MyD88 and IKK-alpha/beta, and modulates NF-kappaB signaling in macrophages. In the present study, we found that ectopic expression of STAP-2 inhibited Epstein-Barr virus (EBV) LMP1-mediated NF-kappaB signaling and interleukin-6 expression. Indeed, STAP-2 associated with LMP1 through its PH and SH-2-like domains, and these proteins interacted with each other in EBV-positive human B cells. We further found that STAP-2 regulated LMP1-mediated NF-kappaB signaling through direct or indirect interactions with tumor necrosis factor receptor (TNFR)-associated factor (TRAF) 3 and TNFR-associated death domain protein (TRADD). STAP-2 mRNA was induced by expression of LMP1 in human B cells. Furthermore, transient expression of STAP-2 in EBV-positive human B cells decreased cell growth. Finally, STAP-2 knockout mouse embryonic fibroblasts showed enhanced LMP1-induced cell growth. These results suggest that STAP-2 acts as an endogenous negative regulator of EBV LMP1-mediated signaling through TRAF3 and TRADD.