Significance Regulated activation of the NF-κB family of transcription factors is important for normal development, immune cell function, and inflammatory responses. NEMO, the NF-κB essential modulator, controls activation of the canonical IKK complex and NF-κB–mediated cellular responses, but details of how this is achieved are not fully known. Our results show that C-terminal mutations in NEMO can cause hyperactivation of inflammatory responses to Toll-like receptor and TNF ligands through impaired recruitment of the negative NF-κB regulator A20/TNFAIP3. Our results help to explain the inflammatory symptoms in patients harboring these NEMO mutations. Furthermore, our findings suggest that targeting this molecular interaction by enhancing A20 expression or its recruitment to the NEMO C-terminus may be a therapeutic strategy for human inflammatory disease.