Post-mitotic neurons in the developing cortex migrate along radial glial fibers to the proper location in the cortical plate and form the layered structure. Here we report that the radial migration of rat layer II/III cortical neurons requires the guidance of an extracellular diffusible factor Semaphorin-3A (Sema3A). Sema3A is expressed in a descending gradient across the cortical layers, whereas its receptor neuropilin-1 (NP1) is expressed at a high level in migrating neurons. Using in utero eletroporation to down-regulate or conditional knockout of NP1 in newborn cortical neurons impeded their radial migration by disrupting their radial orientation during migration without altering their cell fate. Studies in cultured cortical slices further showed the requirement of the endogenous gradient of Sema3A for the proper migration of newborn neurons. Finally, transwell chemotaxis assays showed that isolated newborn neurons were attracted by Sema3A. Thus, Sema3A may serve as a chemoattractive guidance signal for radial migration of newborn cortical neurons toward upper layers.