Pancreatic cancer is a highly malignant disease, with a 5-year survival rate of less than 4%. Thus, new therapies for this deadly disease are urgently required. In this study, we sought to investigate whether combination treatments with gemcitabine and clofarabine result in synergistic cytotoxic effects against human pancreatic cancer cells. The type and extent of cytotoxic interactions between gemcitabine and clofarabine in three human pancreatic cancer cell lines were determined by MTT assays and standard isobologram analysis. The effects of the two agents on cell cycle distribution and apoptosis were determined by flow cytometry. Clofarabine significantly and synergistically enhanced gemcitabine cytotoxicities in all of the cell lines tested. This was accompanied by a nearly complete S phase arrest and synergistic induction of apoptosis (cooperativity index = 0.67). Combined treatment of gemcitabine and clofarabine resulted in synergistic cytotoxicities in vitro. Our results strongly suggest potential clinical benefits for using this drug combination to treat pancreatic cancer patients.