ROPs (Rho-related GTPases of plants) are small GTPases that are plant-specific signaling proteins. They act as molecular switches in a variety of developmental processes. In this study, seven cDNA clones coding for ROP GTPases have been isolated in Medicago truncatula, and conserved and divergent domains are identified in these predicted MtROP proteins. Phylogenetic analysis has indicated that MtROPs are distributed into groups II, III, IV but group I. MtROP genes are expressed in various tissues at different levels. A quantitative reverse transcription PCR analysis indicated that these MtROP genes have different expression profiles in the roots in response to infection with rhizobia. The expression of MtROP3, MtROP5 and MtROP6 are increased, as the expression of Nod factor or rhizobial-induced marker genes—NFP, Rip1 and Enod11; MtROP10 has showed enhanced expression at a certain post-inoculation time point. No significant changes in MtROP7 and MtROP9 expression have been detected and MtROP8 expression is dramatically decreased by about 80%–90%. Additionally, ROP promoter-GUS analysis has showed that MtROP3, MtROP5 and MtROP6 have elevated expression in transgenic root hairs after rhizobial inoculation. These results might suggest a role for some ROP GTPases in the regulation of early stages during rhizobial infection in symbiosis.