Background: Pancreatic cancer is the fourth most common cause of cancer death in the U.S. Gemcitabine is the most active single agent for pancreatic cancer, but chemoresistance to gemcitabine is common. Nuclear factor kappa-B (NF-κB) has been implicated in the chemoresistance of solid organ malignancies. We hypothesized that gemcitabine induces NF-κB activity, and inhibition of NF-κB promotes the antiproliferative effects of gemcitabine on pancreatic cancer. Methods: Panc-1, PaCa-2, and BxPC-3 human pancreatic cancer cell lines were treated with gemcitabine in the presence/absence of curcumin or LC-1 (novel NF-κB inhibitor). NF-κB DNA binding activity was measured using an electromobility shift assay (EMSA). The expression of IκB-α (endogenous NF-κB inhibitor) was measured using Western blot. Proliferation was determined using trypan blue-excluded cell counts. Results: NF-κB was constitutively active in all pancreatic cancer lines. Gemcitabine treatment induced an increase in NF-κB activity in all lines. In BxPC-3, gemcitabine (0.2μM) increased NF-κB activity at 24 hours by 21%. In Panc-1 and PaCa-2, gemcitabine (0.2-10μM) did not change NF-κB activity at 24 hours; however, at 48 hours it increased NF-κB activity by 67% and 20%, respectively. Individually, gemcitabine (0.005-20μM) and NF-κB targeted agents, curcumin (0.05-10μM) and LC-1 (1-10μM), dose-dependently inhibited proliferation in all lines. The antiproliferative effects of gemcitabine in combination with curcumin on average were slightly greater than additive. Panc-1 and PaCa-2 were the most sensitive to the combination. LC-1 was synergistic in promoting the antiproliferative effect of gemcitabine in Panc-1 cells. Conversely, BxPC-3 and PaCa-2 failed to show any benefit of the combination with LC-1. In the resistant PaCa-2 cells, LC-1 in combination with gemcitabine decreased NF-κB activity 16% compared to baseline (total 36%). Conversely, in the sensitive Panc-1 cells, LC-1 in combination with gemcitabine decreased NF-κB activity 65% (total 132%). In the sensitive Panc-1 cells, gemcitabine decreased the (30%) level of inhibitory IκB-α (0.2 -20μM) at 24 hours. Conversely, gemcitabine failed to decrease IκB-α in the resistant PaCa-2 (0.2-20μM, 24-72 hr). Conclusion: Gemcitabine induced a variable increase in NF-κB activity in pancreatic cancer cells. Greater gemcitabine-induced increases in NF-κB activity correlated with decreases in IκB-α and a greater antiproliferative effect of the gemcitabine-NF-κB targeted agent combination. NF-κB targeting may improve the effectiveness of gemcitabine in the treatment of patients with pancreatic cancer.