Mechanisms underlying nodulation factor signaling downstream of the nodulation factor receptors (NFRs) have not been fully characterized. In this study， clathrin heavy chain1 (CHC1) was shown to interact with the Rho-Like GTPase ROP6， an interaction partner of NFR5 in Lotus japonicus. The CHC1 gene was found to be expressed constitutively in all plant tissues and induced in Mesorhizobium loti-infected root hairs and nodule primordia. When expressed in leaves of Nicotiana benthamiana， CHC1 and ROP6 were colocalized at the cell circumference and within cytoplasmic punctate structures. In M. loti-infected root hairs， the CHC protein was detected in cytoplasmic punctate structures near the infection pocket along the infection thread membrane and the plasma membrane of the host cells. Transgenic plants expressing the CHC1-Hub domain， a dominant negative effector of clathrin-mediated endocytosis， were found to suppress early nodulation gene expression and impair M. loti infection， resulting in reduced nodulation. Treatment with tyrphostin A23， an inhibitor of clathrin-mediated endocytosis of plasma membrane cargoes， had a similar effect on down-regulation of early nodulation genes. These findings show an important role of clathrin in the leguminous symbiosis with rhizobia.