ADP-ribosylation factors (Arf), a family of small GTP-binding proteins, play essential role in the regulation of housekeeping functions such as cellular trafficking in yeast, mammalian, and plants. Although this family of genes are strictly conserved both functionally and sequentially throughout Eukaryotae, putative lineage-specific members of these genes have been reported to be sub-or neofunctionalized in plant kingdom. In this study, we investigated the possible lineage-specific functional evolution of four Arf1 paralogous genes in the development and maintenance of nodular structures of Medicago truncatula. Even though all of MtArf1 paralogs were shown to have certain amount of expression in every tissue of M. truncatula, the expression of these Arf1 paralogs was significantly variable and higher in nodular tissues. To better gage the functional evolution of paralogous genes, the spatiotemporal expressional profiles of MtArf1 paralog genes have been investigated by measuring the level of variation in the amount of the transcripts during nodule formation immediately after the infection of roots of Medicago plants with Sinorhizobium melliloti. Of these previously described genes, MtrGB13597775, solely expressed at 72 hpi (hour post inoculation) and the expression level of MtrGB27524313 significantly hiked in the nodules at 6 dpi (days post inoculation) hinting possible subfunctionalization of Arf paralogs. To get more evidence about the possibility of nodule-specific functionilization of MtArf1 paralogs, the level of expression of these genes was also checked at protein level by Western blot analyses. Arf1 protein level was much higher in the infected roots and nodules in comparison to shoots. The findings in this study could suggest that Arf1 genes likely to play an important role in the nodule formation and maintenance in M. truncatula and the possibility of functional specialization of the duplicated Arf genes.