Although Y14 is known to be a component of the exon junction complex, we previously reported that Y14 regulates IL-6-induced STAT3 activation. In this study, we showed that endogenous Y14 positively regulated TNF-alpha-induced IL-6 expression in HeLa cells. Small interfering RNA-mediated Y14-knockdown reduced TNF-alpha-induced and NF-kappaB-mediated transcriptional activity, phosphorylation/degradation of IkappaBalpha, and nuclear localization of NF-kappaB/p65. As in the case of IL-6 stimuli, Y14 enhanced TNF-alpha-induced STAT3 phosphorylation, which is important for its nuclear retention. However, our manipulation of Y14 expression indicated that it is involved in TNF-alpha-induced IL-6 expression via both STAT3-dependent and -independent mechanisms. We screened signaling molecules in the TNF-alpha-NF-kappaB pathway and found that Y14 endogenously associated with receptor-interacting protein 1 (RIP1) and TNFR-associated death domain (TRADD). Overexpression of RIP1, but not TRADD, restored TNF-alpha-induced NF-kappaB activation in Y14-knockdown cells, and Y14 overexpression restored TNF-alpha-induced NF-kappaB activation in TRADD-knockdown cells, but not in RIP1-knockdown cells, indicating that Y14 lies downstream of TRADD and upstream of RIP1. Of importance, Y14 significantly enhanced the binding between RIP1 and TRADD, and this is a possible new mechanism for Y14-mediated modification of TNF-alpha signals. Although Y14 associates with MAGOH in the exon junction complex, Y14's actions in the TNF-alpha-NF-kappaB pathway are unlikely to require MAGOH. Therefore, Y14 positively regulates signals for TNF-alpha-induced IL-6 production at multiple steps beyond an exon junction complex protein.