K-ras is a member of ras gene family which is involved in cell survival, proliferation and differentiation. When a mutation occurs in ras gene, the activation of Ras proteins may be prolonged to induce oncogenesis. However, the relationship between K-ras mutation and clinical outcomes in pancreatic cancer patients treated with chemotherapy agents is still under debate. In this study, we constructed five pAcGFP1-C3 plasmids for different types of K-ras gene (WT, G12V, G12R, G12D, and G13D) and stably transfected human pancreatic cancer Bxpc-3 cells with these genes. The wild type and mutant clones showed a comparable growth and expression of K-Ras-GFP fusion protein. The expression of some K-ras mutations resulted in a reduced sensitivity to gefitinib, 5-FU, docetaxel and gemcitabine, while showed no effects on erlotinib or cisplatin. Moreover, compared with the wild type clone, K-Ras downstream signals (phospho-Akt and/or phospho-Erk) were increased in K-ras mutant clones. Interestingly, different types of K-ras mutation had non-identical K-Ras downstream signal activities and drug responses. Our results are the first to reveal the relationship between different K-ras mutation and drug sensitivities of these anti-cancer drugs in pancreatic cancer cells in vitro.