Neural stem cells (NSCs) derived from induced pluripotent stem cells (iPSCs) are becoming an appealing source of cell-based therapies of brain diseases. As such, it is important to understand the molecular mechanisms that regulate the differentiation of iPSCs toward NSCs. It is well known that Notch signaling governs the retention of stem cell features and drives stem cells fate. However, further studies are required to investigate the role of Notch signaling in the NSCs differentiation of iPSCs. In this study, we successfully generated NSCs from human iPSCs using serum-free medium supplemented with retinoic acid (RA) in vitro. We then assessed changes in the expression of Notch signaling-related molecules and some miRNAs (9, 34a, 200b), which exert their regulation by targeting Notch signaling. Moreover, we used a γ-secretase inhibitor (DAPT) to disturb Notch signaling. Data revealed that the levels of the Notch signaling-related molecules decreased, whereas those miRNAs increased, during this differentiation process. Inhibition of Notch signaling accelerated the formation of the neural rosette structures and the expression of NSC and mature neurocyte marker genes. This suggests that Notch signaling negatively regulated the neuralization of human iPSCs, and that this process may be regulated by some miRNAs.