AtNoa1/Rif1 (formerly referred to as AtNos1) has been shown to modulate nitric oxide (NO) content in Arabidopsis. As NO generation in the legume-rhizobium symbiosis has been shown, the involvement of an AtNoa1/Rif1 orthologue from Medicago truncatula (MtNoa1/Rif1) during its symbiotic interaction with Sinorhizobium meliloti has been studied. The expression of MtNoa1/Rif1 appeared to occur mainly in nodule vascular bundles and the meristematic zone. Using an RNA interference strategy, transgenic roots exhibiting a significantly decreased level of MtNoa1/Rif1 expression were analysed. NO production was assessed using a fluorescent probe, and the symbiotic capacities of the composite plants upon infection with Sinorhizobium meliloti were determined. The decrease in MtNoa1/Rif1 expression level resulted in a decrease in NO production in roots, but not in symbiotic nodules, indicating a different regulation of NO synthesis in these organs. However, it significantly lowered the nodule number and the nitrogen fixation capacity of the functional nodules. Although having no influence on NO production in nodules, MtNOA1/RIF1 significantly affected the establishment and the functioning of the symbiotic interaction. The impairment of plastid functioning may explain this phenotype.