Transforming growth factor-alpha-activated kinase 1 (TAK1) has been widely recognized as a kinase that regulates multiple intracellular signaling pathways evoked by cytokines and immune receptor activation. We have recently reported that tumor necrosis factor-alpha (TNF-alpha) triggers internalization of epidermal growth factor receptor (EGFR) through a TAK1-p38alpha signaling pathway, which results in a transient suppression of the EGFR. In the present study, we investigated the pathway of intracellular signaling in the opposite direction. Ligand-induced activation of EGFR caused phosphorylation of the TAK1-binding proteins TAB1 and TAB2 in a TAK1-independent manner. EGFR-mediated phosphorylation of TAB1 was completely inhibited by a chemical inhibitor and siRNA of p38alpha. The phosphorylation of TAB1 was occurred at Ser-423 and Thr-431, the residues underlying the p38-mediated feedback inhibition of TAK1. In contrast, phosphorylation of TAB2 was sustained, and largely resistant to p38 inhibition. The inducible phosphorylation of TAB1 interfered with a response of EGF-treated cells to TNF-alpha-induced TAK1 activation, which led to the reduction of NF-kappaB activation. Collectively, these results demonstrated that EGFR activation interfered with TNF-alpha-induced TAK1 activation via p38-mediated phosphorylation of TAB1.