The NF-?B pathway is regulated by SUMOylation at least at three levels: the inhibitory molecule I?Ba, the IKK subunit ?/NEMO and the p52 precursor p100. Here we investigate the role of SUMO-2/3 in the degradation of I?Ba and activation of NF-?B mediated by TNFa. We found that under conditions of deficient SUMOylation, an important delay in both TNFa-mediated proteolysis of I?Ba and NF-?B dependent transcription occurs. In vitro and ex vivo approaches, including the use of ubiquitin-traps (TUBEs), revealed the formation of chains on I?Ba containing SUMO-2/3 and ubiquitin after TNFa stimulation. The integration of SUMO-2/3 appears to promote the formation of ubiquitin chains on I?Ba after activation of the TNFa signalling pathway. Furthermore, heterologous chains of SUMO-2/3 and ubiquitin promote a more efficient degradation of I?Ba by the 26S proteasome in vitro compared to chains of either SUMO-2/3 or ubiquitin alone. Consistently, Ubc9 silencing reduced the capture of I?Ba modified with SUMO-ubiquitin hybrid chains that display a defective proteasome-mediated degradation. Thus, hybrid SUMO-2/3-ubiquitin chains increase the susceptibility of modified I?Ba to the action of 26S proteasome, contributing to the optimal control of NF-?B activity after TNFa-stimulation.