Homocitrate is a component of the iron-molybdenum cofactor in nitrogenase, where nitrogen fixation occurs. NifV, which encodes homocitrate synthase (HCS), has been identified from various diazotrophs but is not present in most rhizobial species that perform efficient nitrogen fixation only in symbiotic association with legumes. Here we show that the FEN1 gene of a model legume, Lotus japonicus, overcomes the lack of NifV in rhizobia for symbiotic nitrogen fixation. A Fix(-) (non-fixing) plant mutant, fen1, forms morphologically normal but ineffective nodules. The causal gene, FEN1, was shown to encode HCS by its ability to complement a HCS-defective mutant of Saccharomyces cerevisiae. Homocitrate was present abundantly in wild-type nodules but was absent from ineffective fen1 nodules. Inoculation with Mesorhizobium loti carrying FEN1 or Azotobacter vinelandii NifV rescued the defect in nitrogen-fixing activity of the fen1 nodules. Exogenous supply of homocitrate also recovered the nitrogen-fixing activity of the fen1 nodules through de novo nitrogenase synthesis in the rhizobial bacteroids. These results indicate that homocitrate derived from the host plant cells is essential for the efficient and continuing synthesis of the nitrogenase system in endosymbionts, and thus provide a molecular basis for the complementary and indispensable partnership between legumes and rhizobia in symbiotic nitrogen fixation.