The cytokines TNFα and IL-1β induce inflammation through activation of transcription factors NF-κB. TAB2 is an adapter protein that facilitates TNFα and IL-1β-mediated NF-κB activation. In this work, using yeast two-hybrid system TAB2 was identified to interact with NUMBL. The interaction was further confirmed in vitro and in vivo. PTB domain of NUMBL and C-terminal region are required for their interaction. Overexpression of NUMBL inhibited TNFα, IL-1β-induced activation of NF-κB signaling pathway. NUMBL also inhibited TAB2, TAK1, TRAF6 and RIP-induced activation of NF-κB in a dose-dependent manner. We found that NUMBL can impair TAB2 binding to TRAF6 or RIP and inhibit ubiquitination of TRAF6 enhanced by TAB2. Taken together, our data suggest that NUMBL is involved in negative regulation of NF-κB signaling through its interaction with TAB2. These findings also reveal the new functions of NUMBL and implicate that NUMBL potentially links Notch pathway to NF-κB pathway.