OX40 is a member of the tumor necrosis factor receptor (TNF-R) superfamily. We observed that overexpression of OX40 activated NF-κB, which was inhibited by dominant negative forms of TRAF2, NF-κB-inducing kinase (NIK), and IkappaB kinase (IKK) α. This indicates that OX40 signaling leads to NF-κB activation through the same cascade as TNF-R2. We then investigated the negative regulatory function of TRAF3 on OX40-induced NF-κB activation. TRAF3 blocked OX40-, TRAF2-induced NF-κB activation, but not NIK- and IKKα-induced NF-κB activation, indicating that TRAF3 blocks the pathway between TRAF2 and NIK. C-terminal deletion mutants as well as the N-terminal deletion mutant of TRAF3 inhibited NF-κB activation induced by OX40 or TRAF2. Since TRAF3 bound to OX40 through the C-terminal TRAF domain, the C-terminal domain is likely to work as a dominant negative mutant to compete the recruitment of TRAF2 to the receptor, which transmits the signal from OX40 to the downstream, NIK kinase. On the other hand, the N-terminal domain of TRAF3 seems to affect the downstream of TRAF2 binding. Thus, it is suggested that TRAF3 actively inhibits NF-κB activation induced by OX40.