Ubiquitination and deubiquitination of receptor-interacting protein 1 (RIP1) play an important role in the positive and negative regulation of the tumor necrosis factor alpha (TNFalpha)-induced nuclear factor kappaB (NF-kappaB) activation. Using a combination of functional genomic and proteomic approaches, we have identified ubiquitin-specific peptidase 21 (USP21) as a deubiquitinase for RIP1. USP21 is constitutively associated with RIP1 and deubiquitinates RIP1 in vitro and in vivo. Notably, knockdown of USP21 in HeLa cells enhances TNFalpha-induced RIP1 ubiquitination, IkappaB kinase beta (IKKbeta), and NF-kappaB phosphorylation, inhibitor of NF-kappaB alpha (IkappaB alpha) phosphorylation and ubiquitination, as well as NF-kappaB-dependent gene expression. Therefore, our results demonstrate that USP21 plays an important role in the down-regulation of TNFalpha-induced NF-kappaB activation through deubiquitinating RIP1.