Radial migration of excitatory neurons in an inside-out manner is a critical step for the development of a six-layered neocortical structure, defects of which lead to various brain disorders such as lissencephaly. Subplate neurons (SpNs) and Cajal-Retzius cells (CRs) are first-born neurons in the neocortex, both of which dramatically reduce their number after corticogenesis is complete. Although it is well known that CRs play critical roles in the inside-out layering of excitatory neurons by secreting reelin, little is known about the early roles of SpNs except for their contribution to the development of connections between the thalamus and the cortex. Recently we found that SpNs transiently form glutamatergic synapses on multipolar neurons (MpNs), in which synaptic transmission induces multipolar-to-bipolar transition and initiation of locomotion. Blockade of neuronal activity and vesicular release of SpNs led to failure in the initiation of locomotion by MpNs, resulting in their accumulation below the SP. Similar defects were observed after migrating neuron-specific ablation of NMDA receptors (NMDARs) and knockdown of PSD95. On the contrary, local application of glutamate below the SP stimulated locomotion by MpNs. These results revealed a novel developmental role of synapses regulating the timing of the transition of neuronal polarity and the migration mode. As the SP layer is unique to mammals, the mechanism found here might have contributed to the evolution of the neocortical six-layer structure, from which our sophisticated brain activities originated.