Inactivation of survival pathways such as NF-κB, cyclooxygenase (COX-2), or epidermal growth factor receptor (EGFR) signaling individually may not be sufficient for the treatment of advanced pancreatic cancer (PC) as suggested by recent clinical trials. 3,3′-Diindolylmethane (B-DIM) is an inhibitor of NF-κB and COX-2 and is a well-known chemopreventive agent. We hypothesized that the inhibition of NF-κB and COX-2 by B-DIM concurrently with the inhibition of EGFR by erlotinib will potentiate the anti-tumor effects of cytotoxic drug gemcitabine, which has been tested both in vitro and in vivo. Inhibition of viable cells in seven PC cell lines treated with B-DIM, erlotinib, or gemcitabine alone or their combinations was evaluated using 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Significant inhibition in cell viability was observed in PC cells expressing high levels of COX-2, EGFR, and NF-κB proteins. The observed inhibition was associated with an increase in apoptosis as assessed by ELISA. A significant down-regulation in the expression of COX-2, NF-κB, and EGFR in BxPC-3, COLO-357, and HPAC cells was observed, suggesting that simultaneous targeting of EGFR, NF-κB, and COX-2 is more effective than targeting either signaling pathway separately. Our in vitro results were further supported by in vivo studies showing that B-DIM in combination with erlotinib and gemcitabine was significantly more effective than individual agents. Based on our preclinical in vitro and in vivo results, we conclude that this multi-targeted combination could be developed for the treatment of PC patients whose tumors express high levels of COX-2, EGFR, and NF-κB. J. Cell. Biochem. 110: 171–181, 2010. © 2010 Wiley-Liss, Inc.