Previous studies have shown biological activity of thymoquinone, an active compound extracted from Nigella sativa, in pancreatic cancer cells; however, preclinical animal studies are lacking. Here, we report, for the first time, the chemosensitizing effect of thymoquinone to conventional chemotherapeutic agents both in vitro and in vivo using an orthotopic model of pancreatic cancer. In vitro studies revealed that preexposure of cells with thymoquinone (25 mumol/L) for 48 h followed by gemcitabine or oxaliplatin resulted in 60% to 80% growth inhibition compared with 15% to 25% when gemcitabine or oxaliplatin was used alone. Moreover, we found that thymoquinone could potentiate the killing of pancreatic cancer cells induced by chemotherapeutic agents by down-regulation of nuclear factor-kappaB (NF-kappaB), Bcl-2 family, and NF-kappaB-dependent antiapoptotic genes (X-linked inhibitors of apoptosis, survivin, and cyclooxygenase-2). As shown previously by our laboratory, NF-kappaB gets activated on exposure of pancreatic cancer cells to conventional chemotherapeutic agents; interestingly, thymoquinone was able to down-regulate NF-kappaB in vitro, resulting in chemosensitization. In addition to in vitro results, here we show for the first time, that thymoquinone in combination with gemcitabine and/or oxaliplatin is much more effective as an antitumor agent compared with either agent alone. Most importantly, our data also showed that a specific target, such as NF-kappaB, was inactivated in animal tumors pretreated with thymoquinone followed by gemcitabine and/or oxaliplatin. These results provide strong in vivo molecular evidence in support of our hypothesis that thymoquinone could abrogate gemcitabine- or oxaliplatin-induced activation of NF-kappaB, resulting in the chemosensitization of pancreatic tumors to conventional therapeutics.