Flooding induces hypoxia in plant tissues, impacting various physiological and biochemical processes. This study investigates the adaptive response of the roots and nitrogen-fixing nodules of Medicago truncatula in symbiosis with Sinorhizobium meliloti under short-term hypoxia caused by flooding. Four-week-old plants were subjected to flooding for 1-4 days. Physiological parameters as well as the expression of the senescence marker gene MtCP6 remained unchanged after 4 days of flooding, indicating no senescence onset. Hypoxia was evident from the first day, as indicated by the upregulation of hypoxia marker genes (MtADH, MtPDC, MtAlaAT, MtERF73). Nitrogenfixing capacity was unaffected after 1 day but markedly decreased after 4 days, while energy state (ATP/ADP ratio) significantly decreased from 1 day and was more affected in nodules than in roots. Nitric oxide (NO) production increased in roots but decreased in nodules after prolonged flooding. Nitrate reductase (NR) activity and expression of genes associated with Phytoglobin-NO (Pgb-NO) respiration (MtNR1, MtNR2, MtPgb1.1) were upregulated, suggesting a role in maintaining energy metabolism under hypoxia, but the use of M. truncatula nr1 and nr2 mutants, impaired in nitrite production, indicated the involvement of these two genes in ATP regeneration during initial flooding response. The addition of sodium nitroprusside or tungstate revealed that Pgb-NO respiration contributes significantly to ATP regeneration in both roots and nodules under flooding. Altogether, these results highlight the importance of NR1 and Pgb1.1 in the hypoxic response of legume root systems and show that nodules are more sensitive than roots to hypoxia.