Nogo-A is a myelin derived neurite outgrowth inhibitor which is not only highly expressed in mature oligodendrocytes, but also in various kinds of neurons in the central nervous system. However, the function of neuronal Nogo-A remains unclear. To investigate the function of Nogo-A in neurons, human embryonic kidney cell line 293FT was used as a model, along with the pathway profiling reporter system to check the involvement of Nogo-A in regulating the intracellular signaling pathway. It was found that over-expression of Nogo-A could activate NF-κB signaling specifically, then followed by using different Nogo-A alternative splicing isoforms and a serial of truncations, it was confirmed that the activation of NF-κB by Nogo-A relied on its N-terminal proline rich domain. Furthermore, by co-expressing the dominant negative mutants of some NF-κB pathway associated proteins, it revealed that the IκBα,TRAF6, Rac/Cdc42 were involved in Nogo-A inducing activation of NF-κB. Above all, these results indicate that Nogo-A could significantly activate NF-κB, and this activation depended primarily on its N-terminal proline rich domain.