We recently reported the potential of genistein in augmenting gemcitabine-induced killing of pancreatic cancer (Banerjee, S.et al., Cancer Research 2005;65:9064–72). Sincecis-diaminedichloroplatinum (II) (cisplatin) is widely used against solid tumors, we further investigated whether genistein pretreatment could be used as a novel strategy for cisplatin-induced killing of pancreatic cancer cellsin vitroand enhanced antitumor activityin vivo. Ourin vitroresults showed that pretreatment of cells with genistein followed by cisplatin resulted in significant loss of cell viability and potentiated apoptosis irrespective of the metastatic ability of cells. Mechanistically, genistein augmented cisplatin induced killing by down regulating transcription factor-NF-κB and anti-apoptotic Akt expression. NF-κB was found upregulated when pancreatic cancer cells were exposed to cisplatin, suggesting the potential mechanism of acquired chemo-resistance. In addition, we also showed, for the first time, that genistein in combination with cisplatin is more effective antitumor agent in our orthotopic tumor model. But most importantly, our data also showed that a specific target, such as NF-κB, was inactivated in animal tumors treated with genistein and cisplatin. Immunohistochemical data showed reduced staining for phospho-p65, Bcl-xL and MMP-9 in treated tumors compared to control tumors, but the lowest activity was seen in the combination group. These results provide strong molecularin vivoevidence in support of our hypothesis that inactivation of the NF-κB signaling pathway by genistein results in the chemo-sensitization of pancreatic tumors to cisplatin, which is likely to be an important and novel strategy for the treatment of pancreatic cancer. © 2006 Wiley-Liss, Inc.