Pancreatic cancer is a lethal disease as current chemotherapies with gemcitabine (GEM) are still insufficient. Accumulating evidence suggests that cancer stem cells (CSC) are responsible for chemoresistance and that CD133 is one of the CSC markers in pancreatic cancer. Interferon-alpha (IFN-α), a cytokine with pleiotropic effects, has direct cytotoxic and cytostatic effects on tumor cells. The aim of the present study was to investigate whether IFN-α can modulate the chemosensitivity of a human pancreatic cancer cell line, Capan-1, to GEM. Cell cycles were evaluated for response to GEM with and without IFN-α by BrdU assay. GEM inhibited Capan-1 cell growth in a dose-dependent manner. GEM (IC50; 100 ng/mL) treatment reduced the number of both CD133+ and CD133 cells in the S phase, induced apoptosis of CD133 cells more than that of CD133+ cells and increased accumulation of CD133+ cells into the G0/G1 phase. These results infer that CD133+ cells take shelter into the G0/G1 phase from GEM treatment. IFN-α modulated CD133+ cells from the G0/G1 phase to the S phase. Consequently, apoptosis was accelerated in both CD133+ and CD133 cells after IFN-α combined with GEM treatment. Furthermore, GEM combined with IFN-α treatment showed a significant tumor suppressive effect in the in vivo study. Importantly, CD133+ cells showed CSC-like properties, such as generation of spheres, highly invasive ability and high tumorigenesis. These results suggest that IFN-α, as a modulator, could contribute to the treatment of CD133+ cancer cells and be effective in combined chemotherapies with GEM for pancreatic cancer stem-like cells. (Cancer Sci 2012; 103: 889–896)