In the central nervous system, midline cells play a pivotal role for inducing or suppressing axonal midline crossing. Corticospinal and ipsilaterally projecting spinal neurons, which express α-chimaerin, a Rac-specific GTPase activating protein, and EphA4, an upstream molecule of α-chimaerin, show little midline crossing of axons in the spinal cord. We previously reported that in the α-chimaerin mutants, axons expressing EphA4 are no longer able to be repulsive to ephrinB3-positive midline cells and are aberrantly crossing the midline of the spinal cord (Iwasato et al., Cell 2007). In the α-chimaerin mutants, the spinal cords also show abnormal shapes: flattened dorsal funiculus and enlarged dorsomedial gray matter (Iwasato et al., Cell 2007). In the present study, we found that the α-chimaerin mutants show accumulation of EphA4-positive cells in the midline of the dorsal spinal cord, suggesting that α-chimaerin is required to exclude EphA4-positive cells from the midline. The accumulation of EphA4-positive cells may enlarge dorsomedial gray matter and press dorsal funiculus in α-chimaerin mutants. Besides, the wall of ephrinB3-positive cells, continuously arranging on the midline between the dorsal funiculus and lumen in wild type mice, was disconnected in the α-chimaerin mutant mice. The crevice of ephrinB3-positive cell wall may at least partly contribute to aberrant midline crossing of EphA4-positive axons in α-chimaerin mutants. We will discuss the importance of EphA4-α-chimaerin signaling for midline characteristics of the spinal cord and neural circuit formation.