Newborn cortical neurons undergo radial migration from the ventricular zone (VZ) toward the surface of the cortical plate (CP) during the morphogenesis of cerebral cortex. The spatial-temporal signals that control this stereotyped radial migration remain elusive. Here we report that in developing rat cortex, expression of the Slit receptor family member Robo3 in newborn neurons is essential for these neurons to migrate into the CP. We found that the CP entry was prevented when Robo3 but not Robo2 expression was down-regulated by specific siRNAs. This migration defect was conquered when both Robo3 and Robo2 were simultaneously down-regulated, suggesting the emergence of a Robo2-dependent migration retardation in the absence of Robo3 in these neurons. Moreover, cultured cortical neurons with reduced Robo3 expression showed Slit-induced neurite retraction and repulsive migration away from co-cultured Slit-secreting cell aggregates only when Robo2 was present in the neurons. Thus, Robo3 may allow the radially migrating neurons to invade the CP by suppressing Robo2-mediated repulsion in response to CP-derived Slit1.