Nuclear factor κB(NF-κB) is an important cellular transcription factor. The important role of NF-κB-mediated cell signal transduction pathway in apoptosis is a hot topic at home and abroad. In order to discover new regulators in NF-κB signaling pathway, a high-throughput cell-based screening model based on dual luciferase reporters system was established, a number of genes that can activate NF-κB signal pathway were obtained by screening of 439 novel function genes. Among them, TMEM9B can obviously activate NF-κB signaling pathway. Further experiments showed that TMEM9B activated NF-κB signaling pathway in a dose-dependent pattern. Western blotting and EMSA experiments confirmed that TMEM9B can promote the degradation of IκBα (a cytoplasm inhibitor of NF-κB), and cause NF-κB shift from the cytoplasm to nucleus. At the same time, flow cytometry result demonstrated TMEM9B can induce apoptosis in HEK293T and HeLa cells. In short, a stable and effective screening system for NF-κB has been established, through which TMEM9B was identified to be able to significantly activate NF-κB signal transduction pathway and thus cause cells apoptosis.