Background <p>Improved symbiotic nitrogen fixation efficiency between alfalfa (<i>Medicago sativa</i> L.) and rhizobia represents a green development strategy that addresses the demand for high‑quality protein, while also serving as a critical measure for safeguarding China’s food security. Currently, there is limited research on how rhizobium inoculation influences alfalfa growth and development through photosynthesis and respiratory metabolism. Furthermore, studies examining the impact of rhizobium strains with differing symbiotic effectiveness on these metabolic pathways remain scarce.</p> Results <p>The number of effective nodules per plant (7), nitrogenase activity (0.29 µmol·g^− 1·h^− 1), and leghemoglobin content (0.76 mg·g^− 1) of the LL2 inoculation group were significantly higher than those of the QL5 group. The aboveground dry weight (0.59&#xa0;g·10 plants^− 1) of LL2 was also significantly greater than that of both the QL5 inoculation treatment and the uninoculated control. These results demonstrate that rhizobium strain LL2 is an efficient symbiotic match for ' Gannong No.9 ' alfalfa, whereas strain QL5 is an inefficient match. Metabolomic analysis revealed that, in leaves, seven differential metabolites were up-regulated in both photosynthetic and respiratory metabolism. Among these, Adenosine 5’-Diphosphate (ADP) was significantly higher in LL2 than in CK (Control) and QL5. In roots, nine differential metabolites were up-regulated. Among these, four metabolites—3-Phosphoglyceric acid, Uridine-5’-diphosphate-glucose, (2&#xa0;S)-2-Isopropylmalate, and L-Glutamic acid—were present at significantly higher levels in LL2 than in both CK and QL5. Compared to the QL5 group, the LL2 inoculation group resulted in significantly higher contents of ADP in leaves and elevated levels of the root metabolites such as the photosynthetic carbon fixation intermediate 3-Phosphoglyceric acid, the glycosyl donor Uridine-5’-diphosphate-glucose, the respiration and nitrogen metabolism-related compounds (2S)-2-Isopropylmalate and L-Glutamic acid. Additionally, in nodules, the key metabolites trehalose-6-phosphate and alpha-D-glucose-6-phosphate (involved in sugar metabolism and the pentose phosphate pathway) were also significantly elevated Among these, ADP and alpha-D-glucose-6-phosphate participate simultaneously in photosynthetic, respiratory, and symbiotic metabolic pathways; 3-Phosphoglyceric acid is involved in both photosynthetic and symbiotic pathways; while (2S)-2-Isopropylmalate and L-Glutamic acid take part in respiratory and symbiotic pathways.</p> Conclusions <p>Following inoculation with LL2, the levels of key metabolites associated with photosynthesis and respiration underwent systematic changes in the leaves, roots, and nodules of the plants. The enhanced symbiotic nitrogen fixation and plant growth were associated with synergistic changes in the host plant’s photosynthetic carbon metabolism, respiratory energy metabolism, and nitrogen assimilation pathways. The findings of this study suggest potential strategies for enhancing nitrogen accumulation, possibly through modulating the energy balance of the symbiotic system, which could improve nitrogen fixation efficiency and ultimately increase legume yield and quality.</p>