Solid tumors contain a population of cancer stem cells (CSCs), and CD133 is widely used as a CSCs marker. We investigated the differences between CD133+ and CD133– cells from the neuroblastoma cell line SH-SY5Y in terms of the expressions of voltage-gated ion channels. A CD133+ enriched (>60%) population was isolated, and a subsequent whole-cell voltage-clamp study showed that these cells predominantly express TEA-sensitive outward K+ currents (IK,TEA) and TTX-sensitive voltage-gated inward Na+ currents (INa). Cell-attached single channel recordings demonstrated higher density of large-conductance (155 pS) channel in CD133+ cells than in CD133– cells. The TEA-sensitivity and single channel conductance indicated the large-conductance Ca2+-activated K+ channels (BKCa). Furthermore, RT-PCR analysis of 22 transcripts of voltage-gated ion channels in SH-SY5Y cells showed the expressions of Cav1.3, Kir2.1, Kv1.4, Kv2.1, Kv4.2, Kv7.1, BKCa, and Nav1.7, and those of BKCa and Nav1.7 were higher in CD133+ than in CD133– cells. In addition, the increase of cytosolic Ca2+ concentration ([Ca2+]c) in response to ionomycin (a Ca2+ ionophore) was higher and more sustained in CD133+ than in CD133– cells. Plausibly membrane hyperpolarization via BKCa might be responsible for the augmented Ca2+ influx observed in CD133+ cells. The physiological implications of the differential expression of BKCa and Nav1.7 in CSCs require further investigation.