EF24 is an IKKβ inhibitor (IC50: 72 μM) containing various anti-tumor activities. In this study, a series of EF24 analogs targeting IKKβ were designed and synthesized. Several IKKβ inhibitors with better activities than EF24 were screened out and B3 showed best IKKβ inhibitory (IC50: 6.6 μM). Molecular docking and dynamic simulation experiments further confirmed this inhibitory effect. B3 obviously suppressed the viability of Hela229, A549, SGC-7901 and MGC-803 cells. Then, in SGC-7901 and MGC-803 cells, B3 blocked the NF-κB signal pathway by inhibiting IKKβ phosphorylation, and followed arrested the cell cycle at G2/M phase by suppressing the Cyclin B1 and Cdc2 p34 expression, induced the cell apoptosis by down-regulating Bcl-2 protein and up-regulating cleaved-caspase3. Moreover, B3 significantly reduced tumor growth and suppressed the IKKβ-NF-κB signal pathway in SGC-7901 xenograft model. In total, this study present a potential IKKβ inhibitor as anti-tumor precursor.