Technical Abstract: Bacteria-mediated acquisition of atmospheric dinitrogen by plants serves as a critical nitrogen source in terrestrial ecosystems, and through its key role in agriculture, this phenomenon has shaped the development of human civilizations. Here we show that, paralleling human agriculture, cultivation of fungal crops by leaf-cutter ants involves fertilization from N2-fixing bacteria. Employing acetylene reduction and stable isotope experiments, we demonstrate that N2 fixation occurs in fungus gardens of eight leaf-cutter species and establish that the ants directly benefit from the presence of N2-fixing symbionts. Furthermore, specialized N2-fixers were consistently isolated from fungus gardens of 80 leaf-cutter ant colonies. The discovery of N2 fixation within the leaf-cutter ant-microbe symbiosis reveals an unrecognized nitrogen source in neotropical ecosystems and highlights the importance of symbiotic N2-fixers for sustainable agriculture.