Misregulation of NF-kappaB signaling leads to infectious, inflammatory, or autoimmune disorders. IkappaB kinase beta (IKKbeta) is an essential activator of NF-kappaB and is known to phosphorylate the NF-kappaB inhibitor, IkappaBalpha, allowing it to undergo ubiquitin-mediated proteasomal degradation. However, beyond IkappaBalpha, few additional IKKbeta substrates have been identified. Here we utilize a peptide library and bioinformatic approach to predict likely substrates of IKKbeta. This approach predicted Ser381 of the K63 deubiquitinase A20 as a likely site of IKKbeta phosphorylation. While A20 is a known negative regulator of innate immune signaling pathways, the mechanisms regulating the activity of A20 are poorly understood. We show that IKKbeta phosphorylates A20 in vitro and in vivo at serine 381, and we further show that this phosphorylation event increases the ability of A20 to inhibit the NF-kappaB signaling pathway. Phosphorylation of A20 by IKKbeta thus represents part of a novel feedback loop that regulates the duration of NF-kappaB signaling following activation of innate immune signaling pathways.