To date, no therapy has been found to which pancreatic cancer responds with the exception of surgical resection in early stages. Recently, gemcitabine has become the standard of care for chemotherapy in those patients with advanced disease. Most pancreatic tumors however, develop resistance to gemcitabine. The aim of this study is to clarify the mechanism of resistance to gemcitabine in human pancreatic cells. Using a cell selection method, a human pancreatic cancer cell line resistant to gemcitabine was established. Cellular proliferation and viability were determined by MTT assay. The cell line with acquired resistance was also found to have cross resistance to fluorouracil. Brefeldin-A (BFA) has been used as a tool for studies of intracellular protein traffic, rather than as an anticancer drug. BFA displays the same effects on wild type cells and those with acquired resistance. Gemcitabine combined with BFA in low doses is significantly more effective than gemcitabine alone against MIA PaCa-2 cell line. Our data suggest that the gemcitabine-resistant and 5-FU-resistant pathways may partially overlap each other. In short, BFA may be used as a modulator of gemcitabine.