Nuclear factor-κB (NF-κB) and Akt are two major cell-survival pathways that are often constitutively activated in cancer cells. It has been established that these two pathways contribute substantially toward the chemoresistance of cancer cells. Our previous study has demonstrated that NF-κB and Akt cooperatively blunt cytotoxicity induced by cisplatin or etopside in different types of cancer cells in vitro, indicating that the concurrent blocking of these pathways may effectively improve the anticancer efficacy of anticancer therapeutics. In this study, we further investigated the effect of concurrent blockade of NF-κB and Akt on the anticancer activity of cisplatin in vivo in a xenograft tumor model. The NF-κB and Akt pathways in the A549 lung cancer cells were blocked individually or concurrently by the stable transfection of plasmids expressing short hairpin RNAs that target Akt1 and IκB kinase β. The resultant cells with concurrent NF-κB and Akt blockade were significantly more sensitive to cisplatin-induced cell death in vitro. Consistently, tumors derived from cells with the concurrent blockade of NF-κB and Akt were much more sensitive to cisplatin compared with those derived from cells with individual blockage of NF-κB or Akt in a nude mouse xenograft tumor model. Apoptosis was significantly enhanced in the double pathway-suppressed and cisplatin-treated tumors. These results show for the first time that the concurrent blockage of the NF-κB and Akt pathways cooperatively potentiates the antitumor activity of cisplatin in vivo, indicating that this strategy may be potentially useful for clinical anticancer therapy.