Neural recognition molecule NB-3 is involved in neural development and synapse formation. However, its role in axon tract formation is unclear. In this study, we found that the temporal expression of NB-3 in the deep layers of the motor cortex in mice was coincident with the development of the corticospinal tract (CST). Clear NB-3 immunoreactivity in the CST trajectory strongly suggested that NB-3 was expressed specifically in projecting CST axons. By tracing CST axons in NB-3−/− mice at different developmental stages, we found that these axons were capable of projecting and forming a normal trajectory. However, the projection was greatly delayed in NB-3−/− mice compared with wild-type (WT) mice from the embryonic to postnatal stages, a period that is coincident with the completion of the CST projection in mice. Subsequently, although their projection was delayed, CST axons in NB-3−/− mice gradually completed a normal projection. By stage P21, the characteristics of CST projections in NB-3−/− mice were not statistically different from those in WT mice. In addition, we found that the branching of CST axons into spinal gray matter also was delayed in NB-3−/− mice. The CST innervation area in the spinal gray matter of NB-3−/− mice was greatly reduced in comparison with WT mice until P30 and gradually became normal by P45. These data suggest that NB-3 is involved in the normal projection and terminal branching of developing CST axons. J. Comp. Neurol. 520:1227–1245, 2012. © 2011 Wiley Periodicals, Inc.