The principal objective of this study was to evaluate the chemopreventive and therapeutic effects of a combination of all-trans-retinoic acid (RA) and knockdown of delta-like 1 homologue (Drosophila) (DLK1) on neuroblastoma, the most common malignant disease in children. As unfavorable neuroblastoma is poorly differentiated, neuroblastoma cell was induced differentiation by RA or DLK1 knockdown. Neuroblastoma cells showed elongated neurite growth, a hallmark of neuronal differentiation at various doses of RA, as well as by DLK1 knockdown. In order to determine whether or not a combination of RA and DLK1 knockdown exerts a greater chemotherapeutic effect on neuroblastoma, cells were incubated at 10 nM RA after being transfected with SiRNA-DLK1. Neuronal differentiation was increased more by a combination of RA and DLK1 knockdown than by single treatment. Additionally, in order to assess the signal pathway of neuroblastoma differentiation induced by RA and DLK1 knockdown, treatment with the specific MEK/ERK inhibitors, U0126 and PD 98059, was applied to differentiated neuroblastoma cells. Differentiation induced by RA and DLK1 knockdown increased ERK phosphorylation. The MEK/ERK inhibitor U0126 completely inhibited neuronal differentiation induced by both RA and DLK1 knockdown, whereas PD98059 partially blocked neuronal differentiation. After the withdrawal of inhibitors, cellular differentiation was fully recovered. This study is, to the best of our knowledge, the first to demonstrate that the specific inhibitors of the MEK/ERK pathway, U0126 and PD98059, exert differential effects on the ERK phosphorylation induced by RA or DLK1 knockdown. Based on the observations of this study, it can be concluded that a combination of RA and DLK1 knockdown increases neuronal differentiation for the control of the malignant growth of human neuroblastomas, and also that both MEK1 and MEK2 are required for the differentiation induced by RA and DLK1 knockdown.