Rhizobia induces nitrogen- fi xing nodules in legumes used in agricultural production, providing a direct source of combined nitrogen to leguminous crops. Small peptides, such as CLAVATA3/EMBRYO SURROUNDING REGION peptides (CLE), are known to regulate the formation and development of nitrogen- fi xing nodules in legumes. Root meristem growth factor (RGF) peptides from Medicago truncatula not only regulate root development but also modulate nodulation symbiosis with Sinorhizobium meliloti . However, the impact of RGF peptides from one leguminous species on the others remains unclear. In this study, we investigate the effects of the RGF family peptide MtRGF6p from M. truncatula on nodulation symbiosis and root development in Lotus japonicus . The MtRGF6 gene is predominantly expressed in the root nodules of M. truncatula and shows low identity with RGF homologous genes from L. japonicus . The gene promoter is active in the primordia of root nodules and lateral roots, as well as in young nodules and roots, and the meristem, infection, and nitrogen- fi xing regions of the mature nodule. Chemically synthesized MtRGF6p promoted primary root growth in M. truncatula but suppressed the growth of L. japonicus primary roots. The peptide negatively affected the initiation of nodule primordia, the formation of infection threads, and nodulation in both legumes, with a low dosage showing effects on L. japonicus compared to M. truncatula . These results suggest that the MtRGF6 peptide from M. truncatula may serve as an inter-species signal affecting the root organ development of L. japonicus .