A20 negatively regulates inflammation by inhibiting the nuclear factor kappaB (NF-kappaB) transcription factor in the tumor necrosis factor-receptor (TNFR) and Toll-like receptor (TLR) pathways. A20 contains deubiquitinase and E3 ligase domains and thus has been proposed to function as a ubiquitin-editing enzyme downstream of TNFR1 by inactivating ubiquitinated RIP1. However, it remains unclear how A20 terminates NF-kappaB signaling downstream of TLRs. We have shown that A20 inhibited the E3 ligase activities of TRAF6, TRAF2, and cIAP1 by antagonizing interactions with the E2 ubiquitin conjugating enzymes Ubc13 and UbcH5c. A20, together with the regulatory molecule TAX1BP1, interacted with Ubc13 and UbcH5c and triggered their ubiquitination and proteasome-dependent degradation. These findings suggest mechanism of A20 action in the inhibition of inflammatory signaling pathways.