Cancer stem cell characteristics, especially their self-renewal and clonogenic potentials, play an essential role in malignant progression and response to anticancer therapies. Currently, it remains largely unknown what pathways are involved in the regulation of cancer cell stemness and differentiation. Previously, we found that delta-like 1 homolog (Drosophila) or DLK1, a developmentally regulated gene, plays a critical role in the regulation of differentiation, self-renewal, and tumorigenic growth of neuroblastoma cells. Here, we show that DLK1 specifically interacts with the prohibitin 1 (PHB1) and PHB2, two closely related genes with pleiotropic functions, including regulation of mitochondrial function and gene transcription.DLK1interacts with thePHB1-PHB2complex via its cytoplasmic domain and regulates mitochondrial functions, including mitochondrial membrane potential and production of reactive oxygen species. We have further found that PHB1 and especially PHB2 regulate cancer cell self-renewal as well as their clonogenic potential. Hence, the DLK1-PHB interaction constitutes a new signaling pathway that maintains clonogenicity and self-renewal potential of cancer cells.