PRAS40 has been shown to have a crucial role in the repression of mammalian target of rapamycin (mTOR). Nonetheless, PRAS40 appears to have an oncogenic function in cancer cells. Whether PRAS40 mediates signaling independent of mTOR inhibition in cancer cells remains elusive. Here PRAS40 overexpression in lung adenocarcinoma and cutaneous melanoma was significantly correlated to worse prognosis. And we identified an unexpected role for PRAS40 in the regulation of nuclear factor (NF)-κB signaling. P65, a subunit of the NF-κB transcription factor complex, was confirmed to associate with PRAS40 by glutathione S-transferase co-precipitation. Importantly, we found that PRAS40 can enhance NF-κB transcriptional activity in a manner dependent upon PRAS40-P65 association. Furthermore, we found that a small p65-derived peptide can disrupt the PRAS40-P65 association and significantly decrease NF-κB transcriptional activity. These findings may help elucidate the pleiotropic functions of PRAS40 in cells and suggest a novel therapeutic strategy in cancer patients with high expression of PRAS40 and NF-κB.