Bacteria carefully regulate metabolic processes to efficiently colonize and persist within the human host. One mechanism bacteria employ is gene regulation. In Escherichia coli, the catabolism of sialic acid is controlled by the GntR-type transcriptional regulator NanR, but the mechanism of gene regulation is unknown. We first demonstrate that NanR binds as a dimer to a total of three GGTATA direct repeats that make up the DNA recognition site, forming a hexameric assembly. Interestingly, we found this binding is cooperative and demonstrate it is mediated by a unique N-terminal extension, likely through protein-protein interactions. To understand how DNA-binding is attenuated by the effector, we solve the co-crystal structure of E. coli NanR in the presence of N-acetylneuraminic acid to 2.1 Å, supporting the hypothesis that this is the effector molecule, and identify a metal-binding motif that coordinates zinc. The structure is asymmetrical with N-acetylneuraminic acid and zinc only present in one monomer, informing the molecular details of the conformational change that occurs following binding to attenuate DNA-binding activity. Notably, we report the structure of the NanR-DNA hetero-complex to 3.9 Å using cryo-electron microscopy, and the first structural evidence of a multimeric assembly process within the GntR superfamily. Together, our results give the first molecular insight into the mechanism of the NanR-DNA interaction in E. coli, which enhances our understanding of sialic acid gene regulation in bacteria.