Repulsive guidance molecule a (RGMa), which binds to its receptor neogenin, has been well determined as a repulsive axon guidance molecule. However, whether RGMa affects the growth of hippocampal mossy fibers, the axons of dentate granule cells, has been unknown. In the present study, we found that the primary neurons in the hippocampus express both RGMa and neogenin in the postnatal rats. To examine the role of RGMa in the mossy fiber growth, the morphology of granule cells was clearly visualized by transfecting membrane-targeted green fluorescent protein using a single-cell electroporation method in cultured hippocampal slices. In the slice cultures, we found that intrinsic RGMa is required to inhibit excess branching of the hippocampal mossy fibers. Furthermore, hyperexcitability-induced aberrant branching of the mossy fibers in the cultured slices was blocked by applying the recombinant RGMa protein. Therefore, this study suggests that RGMa regulates the proper axonal branching of hippocampal mossy fibers.