Fibroblast-like synoviocytes (FLSs) of patients with rheumatoid arthritis (RA FLSs) exhibit prosurvival, rather than apoptotic, response to tumor necrosis factor (TNF)-α stimulation. Here, we show that JAB1 is a critical regulator of the TNF-α-mediated anti-apoptosis pathways in RA FLSs. We found that knock-down of JAB1 using small interfering (si)RNA led to restoration of the TNF-α-induced apoptosis response, reduction of nuclear factor-κB activity, delayed degradation of IκB-α, and inhibited phosphorylation of JNK. Analysis of the interactions of JAB1 by reciprocal co-immunoprecipitations and confocal microscopy revealed that JAB1 interacts with TNF receptor-associated-factor 2 (TRAF2). The generation of the anti-apoptotic signal on binding of TNF-α to the TNF receptor (TNFR)1 has been shown to be associated with the recruitment of TRAF2 to the TNFR1 in a process that requires ubiquitination of TRAF2 with lysine-63-linked polyubiquitin chains. We found that TNF-α stimulation of JAB1 siRNA-transfected RA FLSs failed to stimulate ubiquitination of TRAF2. Thus, we conclude that JAB1-regulated ubiquitination of TRAF2 is a novel mechanism whereby TNF-α can induce anti-apoptosis signaling and production of matrix metalloproteinases through activation of nuclear factor-κB and JNK in RA FLSs. Copyright © American Society for Investigative Pathology.