New neurons generated in the adult ventricular-subventricular zone (V-SVZ) migrate rapidly inside the tunnels composed of astrocytes toward the olfactory bulb. We have previously demonstrated that such efficient migration is dependent on the repulsive factor Slit1, secreted by new neurons, which is required for the formation of astrocytic tunnels. During regeneration processes after brain insult, new neurons need to migrate from the V-SVZ toward injured areas through the dense meshwork of activated astrocytes. To visualize the behavior of new neurons migrating in contact with activated astrocytes, we have developed a method for time-lapse imaging of stroke-injured mouse brain slices. Using this system, we found that the speed of neuronal migration is decreased when they reach the dense meshwork of activated astrocytes. The inhibitory effect of astrocytes on neuronal migration was significantly stronger in Slit1 KO mice compared with that in the wild-type, suggesting that Slit1 is involved in neuronal migration through activated astrocytes. Using biosensors visualizing actin polymerization and Cdc42 activity expressed in cultured astrocytes, we found that contact of new neurons transiently suppressed actin polymerization and decreased Cdc42 activation in astrocytes, both of which were dependent on Slit1 from the new neurons. The actin depolymerization in astrocytes induced by neuronal contact was cancelled by expression of constitutively active form of Cdc42 in astrocytes, suggesting that the Slit1-expressing new neurons alter astrocytic morphology through Cdc42 inactivation. Furthermore, Slit1-overexpression in new neurons promoted their migration in association with activated astrocytes in vivo and in vitro. Together, these observations suggest that Slit1-mediated interaction of the new neurons with activated astrocytes facilitates their migration toward the injured area.