The enzyme glucose-1-phosphate thymidylyltransferase (RffH), the product of therffh gene, catalyzes one of the steps in the synthesis of enterobacterial common antigen (ECA), a cell surface glycolipid found in Gram-negative enteric bacteria. InEscherichia coli two gene products, RffH and RmlA, catalyze the same enzymatic reaction and are homologous in sequence; however, they are part of different operons and function in different pathways. We report the crystal structure of RffH bound to deoxythymidine triphosphate (dTTP), the phosphate donor, and Mg2+, refined at 2.6 Å to an R-factor of 22.3% (R free = 28.4%). The crystal structure of RffH shows a tetrameric enzyme best described as a dimer of dimers. Each monomer has an overall α/β fold and consists of two domains, a larger nucleotide binding domain (residues 1–115, 222–291) and a smaller sugar-binding domain (116–221), with the active site located at the domain interface. The Mg2+ ion is coordinated by two conserved aspartates and the α-phosphate of deoxythymidine triphosphate. Its location corresponds well to that in a structurally similar domain of N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU). Analysis of the RffH, RmlA, and GlmU complexes with substrates and products provides an explanation for their different affinities for Mg2+ and leads to a proposal for the dynamics along the reaction pathway.