Myeloid differentiation factor 88 (MyD88) plays a central role in innate immunity response, however, how its activity is tightly regulated remains largely unknown. In this study, we identify MyD88 as a novel substrate of NEDD8, and demonstrate that MyD88 NEDDylation antagonizes its ubiquitination. Interestingly, in response to the stimulation of IL-1 beta, MyD88 NEDDylation is downregulated while its ubiquitination is upregulated. We also show that deNEDDylase NEDP1 serves as a regulator of this process. Furthermore, we demonstrate that NEDD8 negatively regulates the dimerization of MyD88 and suppresses MyD88-dependent NF-kappa B signaling. Taken together, this study reveals that NEDDylation of MyD88 regulates NF-kappa B activity through antagonizing its ubiquitination, suggesting a novel mechanism of modulating NF-kappa B signaling pathway. (C) 2016 Elsevier Inc. All rights reserved.