The corpus callosum (CC) is the biggest commissure that links cerebral hemispheres. Guidepost structures develop in the cortical midline during CC development and express axon guidance molecules that instruct neurons regarding the proper direction of axonal elongation toward and across the cortical midline. Neuropilin-1 (Npn1), a high affinity receptor for class 3 semaphorins (Sema3s) localized on cingulate pioneering axons, plays a crucial role in axon guidance to the midline through interactions with Sema3s. However, it remains unclear which type of Plexin is a component of Sema3 holoreceptors with Npn1 during the guidance of cingulate pioneering axons. To address the role of PlexinA1 in CC development, we examined with immunohistochemistry the localization of PlexinA1, Npn1, and Sema3s using embryonic brains from wild-type (WT) and PlexinA1-deficient (PlexinA1 knock-out (KO)) mice with a BALB/cAJ background. The immunohistochemistry confirmed the expression of PlexinA1 in callosal axons derived from the cingulate and neocortex of the WT mice on embryonic day 17.5 (E17.5) but not in the PlexinA1 KO mice. To examine the role of PlexinA1 in the navigation of callosal axons, the extension of callosal axons toward and across the midline was traced in brains of WT and PlexinA1 KO mice at E17.5. As a result, callosal axons in the PlexinA1 KO brains had a significantly lower incidence of midline crossing at E17.5 compared with the WT brains. To further examine the role of PlexinA1 in CC development, the CC phenotype was examined in PlexinA1 KO mice at postnatal day 0.5 (P0.5). Most of the PlexinA1 KO mice at P0.5 showed agenesis of the CC. These results indicate the crucial involvement of PlexinA1 in the midline crossing of callosal axons during CC development in BALB/cAJ mice.