ABCB1 is a human ABC transporter originally characterized by its ability to cause resistance to chemotherapy drugs in cancer cells, and later found to be functionally expressed in human neural stem/progenitor cells (NSPCs) in vitro. Here, we performed a detailed examination of ABCB1's expression on human NSPCs in vitro and in human fetal brain tissues, and analyzed the cellular properties of the human NSPCs expressing ABCB1. We confirmed that ABCB1 was expressed on the surface of human NSPCs, and its level correlated well with those of Nestin and CD133. The population of fluorescence-activated cell sorter-sorted human NSPCs expressing high levels of ABCB1 showed enrichment of proliferating cells, higher expression of 246 genes (e.g., RGS6, IGFBP7, GFAP, TNC, Hes1), and lower expression of 71 genes (e.g., STMN2, DLX5, BASP1, DCX, CD24) compared with human NSPCs expressing low or no ABCB1. In situ, ABCB1 was selectively expressed in cells in the ventricular or subventricular regions of lateral ventricles that expressed Nestin in human development. These findings suggest that ABCB1 is predominantly expressed in immature human fetal NSPCs in vitro and at early developmental stages in vivo, and that it may be a useful marker for human NSPCs.