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The effects of elevated CO2 (750 ppm vs. 390 ppm) were evaluated on nitrogen (N) acquisition and assimilation by three Medicago truncatula genotypes, including two N-fixing-deficient mutants (dnf1-1 and dnf1-2) and their wild-type (Jemalong). The proportion of N acquisition from atmosphere and soil were quantified by (15)N stable isotope, and N transportation and assimilation-related genes and enzymes were determined by qPCR and biochemical analysis. Elevated CO2 decreased nitrate uptake from soil in all three plant genotypes by down-regulating nitrate reductase (NR), nitrate transporter NRT1.1 and NR activity. Jemalong plant, however, produced more nodules, up-regulated N-fixation-related genes and enhanced percentage of N derived from fixation (%Ndf) to increase foliar N concentration and N content in whole plant (Ntotal Yield) to satisfy the requirement of larger biomass under elevated CO2. In contrast, both dnf1 mutants deficient in N fixation consequently decreased activity of glutamine synthetase/glutamate synthase (GS/GOGAT) and N concentration under elevated CO2. Our results suggest that elevated CO2 is likely to modify N acquisition of M. truncatula by simultaneously increasing N fixation and reducing nitrate uptake from soil. We propose that elevated CO2 causes legumes to rely more on N fixation than on N uptake from soil to satisfy N requirements.