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Fatty acid and central carbon metabolism are crucial energy metabolism reactions. However, to date, few studies have examined their distribution characteristics within the alfalfa–rhizobia symbiotic system. To clarify the distributional differences and accumulation rates of fatty acids and central carbon with this system, we measured the plant phenotype, nodule formation, nitrogen fixation capacity, and key nitrogen metabolism enzyme activities of Medicago sativa ‘Gannong No. 9’ 35 days post-inoculation (dpi) with Sinorhizobia meliloti LL11. Additionally, we employed targeted metabolomics to analyze central carbon and fatty acid metabolites in various tissue samples of symbiotic and control (C.K.) plants, as well as in S. meliloti LL11. We found that plant height; root length; aboveground fresh and dry weights; underground fresh and dry weights; and nitrate reductase, nitrogen reductase, glutamine synthetase, and glutamate synthase activities were significantly higher in the leaves and roots of symbiotic plants than in those of C.K. plants. Compared to symbiotic plants, C.K. plants exhibited higher total central carbon and fatty acid metabolite content, accounting for 38.61% and 48.17% of C.K. plants, respectively. We detected 32 central carbon and 40 fatty acid metabolites in S. meliloti LL11, with succinate (343,180.8603 ng·mL−1) and hexadecanoic acid (4889.7783 ng·mL−1) being the most. In both symbiotic and C.K. plants, central carbon metabolite was considerably higher than the fatty acid metabolite central. Moreover, the carbon metabolites found in symbiotic plants were primarily distributed in pink nodule roots (PNRs), with malate exhibiting the highest content (4,800,612.3450 ng·g−1), accounting for 53.09% of total central carbon metabolite content. Fatty acid metabolites were mainly found in pink root nodules (P.N.s), which are sites of nitrogen fixation. Trans-10-nonadecenoic acid and hexadecanoic acid exhibited the highest contents, comprising >15% of the total fatty acid metabolite content. We found that petroselaidic acid is only present in P.N., which seems to be closely related to the nitrogen fixation reaction in P.N. In general, symbiotic plants transfer central carbon metabolites to nodules via PNRs to drive nitrogen fixation. However, in P.N.s, these metabolites are limited, leading to accumulation in PNRs. Fatty acid metabolites, crucial for nitrogen fixation, are prevalent in P.N.s. Conversely, C.K. plants without nitrogen fixation distribute these metabolites primarily to the stems, emphasizing growth. This study provides new insights into the energy metabolism of symbiotic nitrogen fixation.