NF-B is an important transcription factor involved in various biological responses, including inflammation, cell differentiation, and tumorigenesis. B-Ras was identified as an IB-interacting small GTPase and is reported to disturb cytokine-induced NF-B activation. In this study, we established that B-Ras is a novel type of nuclear-cytoplasmic small GTPase that mainly binds to GTP, and its localization seemed to be regulated by its GTP/GDP-binding state. Unexpectedly, the GDPbinding form of the B-Ras mutant exhibited a more potent inhibitory effect on NF-B activation, and this inhibitory effect seemed to be due to suppression of the transactivation of a p65/ RelA NF-B subunit. B-Ras suppressed phosphorylation at serine 276 on the p65/RelA subunit, resulting in decreased interaction between p65/RelA and the transcriptional coactivator p300. Interestingly, the GDP-bound B-Ras mutant exhibited higher interactive affinity with p65/RelA and inhibited the phosphorylation of p65/RelA more potently than wild-type B-Ras. Taken together, these findings suggest that the GDPbound form of B-Ras in cytoplasm suppresses NF-B activation by inhibiting its transcriptional activation.