Tumor side population (SP) cells display stem like properties that can be modulated by treatment with the calcium channel blocker verapamil. Verapamil can enhance the cytotoxic effects of chemotherapeutic drugs and multi drug resistance by targeting the transport function of the P-glycoprotein (P-gp). This study focused on the therapeutic potential of verapamil on stem-like SP tumor cells, and further investigated its chemosensitizing effects using L3.6pl and AsPC-1 pancreatic carcinoma models. As compared to parental L3.6pl cells (0.9 +/- 0.22%), L3.6pl gemcitabine-resistant cells (L3.6pl(Gres)) showed a significantly higher percentage of SP cells (5.38 +/- 0.99%) as detected by Hoechst 33342/FACS assays. The L3.6pl(Gres) SP cells showed stable gemcitabine resistance, enhanced colony formation ability and increased tumorigenicity. Verapamil effectively inhibited L3.6pl(Gres) and AsPC-1 SP cell proliferation in vitro. A pro-apoptotic effect of verapamil was observed in L3.6pl cells, but not in L3.6pl(Gres) cells, which was linked to their differential expression of P-gp and equilibrative nucleoside transporter-1 (ENT-1). In an orthotopic pancreatic cancer mouse model, both low and high dose verapamil was shown to substantially reduce L3.6pl(Gres)-SP cell tumor growth and metastasis, enhance tumor apoptosis, and reduce microvascular density.