Neuroblastoma (NB) is the most common pediatric malignancy and is considered to possess cancer stem cells (CSCs) properties which can drive tumor initiation and metastasis. β-carotene 15,15'-oxygenase (BCO1) is the main enzyme that catalyzes the first step in vitamin A biosynthesis from pro-vitamin A carotenoids. Retinoids (vitamin A) play a critical role in NB differentiation. However, the biological functions of BCO1 in NB remained to be elucidated. Here, we investigated the effects of BCO1 on NB CSCs with stably expressing BCO1 in NB cells. We show that BCO1 significantly suppressed self-renewal and markers of NB CSCs. Moreover, BCO1 inhibited the metastatic potential of NB cells and suppressed the enzymatic activity and expression of MMPs, as well as expression of HIF-1α and its downstream targets. In vivo, BCO1 reduced the metastatic incidence and volumes of metastatic tumors and downregulated the expression of CSCs markers, MMPs, and HIF-1α in tumor tissues of a mouse xenograft model. A possible mechanism underlying the anti-cancer activities of BCO1 is proposed based on miRNAs sequencing array data which suggests a role for BCO1 in regulating miRNAs associated with neuronal differentiation, cell-cell adhesion, and the Wnt signaling pathway. Thus, our results demonstrate new chemotherapeutic roles for BCO1 in malignant NB that mediate suppression of cancer stemness and metastasis.