The corpus callosum, composed of callosal axons, is the largest structure among commissural connections in eutherian animals. Axon pathfinding of callosal neurons has been shown to be guided by intermediate targets, such as midline glial structures. However, it has not yet been understood completely how axon-axon interactions, another major mechanism for axon pathfinding, are involved in the pathfinding of callosal neurons. Here, we show that callosal axons from the medial and lateral regions of the mouse cerebral cortex pass through the dorsal and ventral parts, respectively, of the corpus callosum. Using an explant culture system, we observed that the axons from the medial and lateral cortices were segregated from each other in vitro, and that this segregation was attenuated by inhibition of EphA3 signaling. We also found that knockdown of EphA3, which is preferentially expressed in the lateral cortex, resulted in disorganized segregation of the callosal axons and disrupted axon pathfinding in vivo. These results together suggest the role of axonal segregation in the corpus callosum, mediated at least in part by EphA3, in correct pathfinding of callosal neurons.