Emerging evidence proposes that most cancers originate from a rare subpopulation of cells, called cancer stem cells (CSCs), which possess characteristics including differentiation, self-renewal, and tumorigenicity. Currently, available therapeutic agents cannot effectively eliminate CSCs. Therefore, the development of a nontoxic, natural treatment that can either overcome chemoresistance or promote the elimination of CSCs is highly desirable. The current study examined whether mulberry leaf (ML) ethanolic extract can effectively eliminate neuroblastoma stem cell-like population. Our data demonstrated that 10-40g/ml of ML extract significantly enhanced differentiation by elongating neurites and reducing clonogenicity and sphere formation as shown by the decreased expression of stem cell markers and increased expression of differentiation markers. The knock-down of delta-like 1 homologue by siRNA enhanced the significant inhibitory effects of 40g/ml of ML extract on colony formation. Furthermore, phosphorylation of the extracellular signal-regulated kinase (ERK) was increased by 20 or 40g/ml of ML extract and the MEK/ERK inhibitors completely blocked differentiation induced by the extract. Taken together, these findings provide experimental evidence that ML may have chemopreventive effects on neuroblastoma cells by inhibiting CSCs characteristics as well as regulating CSCs pathways, which may provide a therapeutic option for controlling the growth of neuroblastoma cells.