Increasing evidence suggests that Rho of plant (ROP) GTPases play important roles in the rhizobium–legume symbiotic nodulation, but the molecular mechanisms of their regulation in symbiosis remain poorly understood. In this study, we showed that ROP4 in Lotus japonicus (LjROP4) is involved in the symbiotic interaction between L. japonicus and Mesorhizobium loti. Tissue expression analysis showed that LjROP4 expressed highly in the root. Histochemical staining analysis showed that after rhizobia inoculation, GUS reporter activity increased in the root vascular bundle, root tip, and lateral root primordia. During nodule development, GUS activity was detected in the cortex of nodule primordia and young nodules. In the mature nodules, GUS activity was detected only in the vascular bundle. Compared with the control, the overexpression of ROP4 and ROP4-CA produced much more pronounced root hair swelling and curling induced by M. loti. The infection event and nodule number noticeably increased, which was consistent with this promotion of root hair deformation. Moreover, RNA interference of LjROP4 produced opposite phenotypes. These data suggest that LjROP4 is required for root hair deformation during rhizobial infection. Thus, our study provides important information about root hair deformation responses induced by nod factors in the early stages of symbiotic interaction.