Tumor necrosis factor alpha (TNF-α) receptor-associated factors (TRAFs) play important roles in TNF-α signaling by interacting with downstream signaling molecules, e.g., mitogen-activated protein kinases (MAPKs). However, TNF-α also signals through reactive oxygen species (ROS)-dependent pathways. The interrelationship between these pathways is unclear; however, a recent study suggested that TRAF4 could bind to the NADPH oxidase subunit p47phox. Here, we investigated the potential interaction between p47phox phosphorylation and TRAF4 binding and their relative roles in acute TNF-α signaling. Exposure of human microvascular endothelial cells (HMEC-1) to TNF-α (100 U/ml; 1 to 60 min) induced rapid (within 5 min) p47phox phosphorylation. This was paralleled by a 2.7- ± 0.5-fold increase in p47phox-TRAF4 association, membrane translocation of p47phox-TRAF4, a 2.3- ± 0.4-fold increase in p47phox-p22phox complex formation, and a 3.2- ± 0.2-fold increase in NADPH-dependent O2− production (all P < 0.05). TRAF4-p47phox binding was accompanied by a progressive increase in extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38MAPK activation, which was inhibited by an O2− scavenger, tiron. TRAF4 predominantly bound the phosphorylated form of p47phox, in a protein kinase C-dependent process. Knockdown of TRAF4 expression using siRNA had no effect on p47phox phosphorylation or binding to p22phox but inhibited TNF-α-induced ERK1/2 activation. In coronary microvascular EC from p47phox−/− mice, TNF-α-induced NADPH oxidase activation, ERK1/2 activation, and cell surface intercellular adhesion molecule 1 (ICAM-1) expression were all inhibited. Thus, both p47phox phosphorylation and TRAF4 are required for acute TNF-α signaling. The increased binding between p47phox and TRAF4 that occurs after p47phox phosphorylation could serve to spatially confine ROS generation from NADPH oxidase and subsequent MAPK activation and cell surface ICAM-1 expression in EC.