The epidermal growth factor receptor (EGFR) is a target of new therapies in most nonhematologic cancers. EGFR blockade alone may not be sufficient for the control of growth and invasion of human pancreas cancer because of the independent activation of Akt and nuclear factor-kappaB (NF-kappaB). The expression of EGFR, Akt, and NF-kappaB was determined in six human pancreatic cancer cell lines. Selected cells for specific expression were treated with erlotinib, genistein, gemcitabine, or the combination. Growth inhibition was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and apoptosis was assayed by ELISA. EGFR, phosphorylated EGFR, phosphorylated Akt, and survivin expressions were determined by immunoblotting. Electrophoretic mobility shift assay was used to evaluate the DNA binding activity of NF-kappaB. Genistein significantly increased (P < 0.05) erlotinib-induced growth inhibition and apoptosis in BxPC-3, CAPAN-2, and AsPC-1 cells. In the BxPC-3 cell line, significant down-regulation of EGFR, phosphorylated Akt, NF-kappaB activation, and survivin was observed in the cells treated with the combination compared with the erlotinib-treated cells. In the HPAC and MIAPaCa cell line, no potentiation of the effects of erlotinib by genistein on cell growth or inhibition of the EGFR/Akt/NF-kappaB was observed. Genistein potentiated growth inhibition and apoptosis of the gemcitabine and erlotinib combination in COLO-357 cell line. Genistein potentiates the growth inhibition and apoptosis induced by erlotinib and gemcitabine in certain pancreatic cancer cells. Akt and NF-kappaB inhibition represents one of the mechanisms for the potentiation of erlotinib- and gemcitabine-induced cell death by genistein.