TRAF6 is an E3 ubiquitin ligase that mediates signaling from members of the tumor necrosis factor and Toll-like receptor superfamilies, including the p75 neurotrophin receptor. Recently, TRAF6 was shown to bind to another p75 cytoplasmic interactor, NRIF, and promote its nuclear localization. Here, we demonstrate that NRIF is a substrate for TRAF6-mediated K63 polyubiquitination and that this modification is necessary for its nuclear translocation. Activation of p75 resulted in NRIF polyubiquitination, association with TRAF6 and nuclear localization. NRIF was polyubiquitinated by TRAF6 in vitro and in cultured cells, and this was abrogated by mutation of K19 in the amino-terminus of NRIF. The K19R mutant NRIF displayed reduced TRAF6 association and neurotrophin-dependent nuclear localization. In neurons from traf6-/- mice, NRIF failed to enter the nucleus in response to p75 activation, and polyubiquitination and nuclear localization were attenuated in traf6-/- brain. Finally, unlike wild-type NRIF, the K19R NRIF failed to reconstitute p75-mediated apoptosis in nrif-/- neurons. These results reveal a unique mechanism of p75 signaling and a novel role for K63-linked ubiquitin chains.