Mutations in Hnf-1α are the most common Mendelian cause of diabetes mellitus. To elucidate the molecular function of a mutational hotspot, we cocrystallized human HNF-1α 83–279 with a high-affinity promoter and solved the structure of the complex. Two identical protein molecules are bound to the promoter. Each contains a homeodomain and a second domain structurally similar to POU-specific domains that was not predicted on the basis of amino acid sequence. Atypical elements in both domains create a stable interface that further distinguishes HNF-1α from other flexible POU-homeodomain proteins. The numerous diabetes-causing mutations in HNF-1α thus identified a previously unrecognized POU domain which was used as a search model to identify additional POU domain proteins in sequence databases.