In in vitro continuous culture, N (neuroblastic)-, S (substrate adherent)- and I (intermediate)-type cells were identified in human neuroblastoma (NB), where I-type is recognized as a stem cell type. Octamer-binding protein 4 (OCT4) is a cell marker used to identify the stemness of cells, whose roles in regulating I-type NB cells have yet to be fully elucidated. In the present study, we assessed the differentiation regulation role of OCT4 in BE (2)-C of I-type cells. We demonstrated that downregulation of OCT4 expression in BE (2)-C was associated with reduced cell proliferation and loss of colony formation ability on soft agar, and prompted BE (2)-C cells to differentiate to S-type cells. By contrast, overexpression of OCT4 increased cell proliferation and colony formation ratio, but no obvious differentiation promotion was observed. Furthermore, induced differentiation of BE (2)-C cells to S-type by 5-bromo-2'-deoxyuridine (BrdUrd) was accompanied by reduced expression of OCT4.