Tumor necrosis factor-associated factor 6 (TRAF6) is an essential adaptor protein for IL-1R or TLR-mediated NF-κB signaling pathway activation. In previous work we have found NUMBL interacts with TAB2 and negatively regulates NF-κB signaling pathway. Here, we report that NUMBL directly binds to TRAF6 in vivo and in vitro. NUMBL down-regulates TRAF6 protein level and shortens its half-life. Furthermore, knockdown of NUMBL significantly increases endogenous TRAF6 protein level in the cultured cortical neurons. In vivo ubiquitination assays indicate that NUMBL promotes the assembly of K48-linked polyubiquitination chains on TRAF6, but has no significant effect on its K63-linked polyubiquitination. Our results collectively reveal that NUMBL interacts with TRAF6 and promotes the degradation of TRAF6 in vivo, leading to the inhibition of NF-κB signaling pathway.