Leguminous plants form nitrogen-fixing root nodules and the number of nodules is controlled by a self-regulating mechanism called autoregulation. However, signaling substances involved in nodule regulation have not been identified. In the present study, we used brassinolide, a most effective molecular species of plant hormone brassinosteroids, and brassinazole, an effective inhibitor of brassinosteroid biosynthesis to determine whether brassinolide played a role in systemic regulation of noduel formation in wild type soybean and its super-nodulating mutant. Foliar application or direct injection of brassinolide into the root base inhibited nodule formation and root development in the super-nodulating mutant (En6500), but not in the parent line (cv. Enrei). The internodes in the plants subjected to foliar application were significantly longer than those in the untreated plants. In contrast, the application of brassinazole on mature leaves or into the culture media resulted in the increase of the nodule number in Enrei. These treatments also inhibited internodal growth in Enrei. The results indicate that brassinosteroids may regulate the nodule number in soybean plants. The function of brassinosteroids for the systemic regulation of nodule formation was examined.