Callosal axons form one of the major axonal tracts in the cerebral cortex, the corpus callosum. During development, callosal axon projections (CAP) depend on not only guidance molecules, but also neuronal activity. We previously showed that reduction of neuronal activity in callosal projection neurons (CPNs) by Kir2.1 overexpression resulted in the disturbance of CAP (Mizuno et al., JNS, 2007). However, little is known about the time window when neuronal activity is critical for the formation of CAP. To investigate this, we employed Tet-off system for time dependent Kir2.1 expression. We expressed Kir2.1 in CPNs under the control of Tet-off gene expression system by in utero electroporation, and turned off the Kir2.1 expression at certain points in development by giving doxycycline to neonatal mice through their mothers. Expressing Kir2.1 throughout development impaired CAP as expected from the previous study. In contrast, suppressing Kir2.1 expression from postnatal 6 to 15 (P6-15) restored CAP. Suppression of Kir2.1 expression in P9-18 or P12-21 did not rescue CAP. These results demonstrate a critical period for a restoration of CAP, and suggest that neuronal activity in P6-15 is critical for CAP. Next, we used channelrhodpsin2 (ChR2) to examine whether optogenetic stimulation can rescue CAP. We expressed ChR2 and Kir2.1 in CPNs, and gave light stimulation by LED attached to the head of mice. Optogenetic stimulation in P11-13 partly recovered CAP. Together, our findings suggest that neuronal activity in the second postnatal week is critical for the formation of CAP. Furthermore, neuronal stimulation by optogenetics may be useful for regeneration of impaired axonal projections.