D-Mannitol has wide applications in food, pharmaceutical, and chemical industries. In this study, we constructed a genetically stable Escherichia coli strain for D-mannitol production by integrating mannitol dehydrogenase (mdh) and fructose permease (fupL) genes of Leuconostoc pseudomesenteroides ATCC 12291 into chromosome of E. coli ATCC 8739 and inactivating other fermentation pathways (including pyruvate formate-lyase, lactate dehydrogenase, fumarate reductase, alcohol dehydrogenase, methylglyoxal synthase and pyruvate oxidase). Using mineral salts medium with glucose and fructose as carbon sources, the engineered strain could produce 1.2 mmol/L D-mannitol after anaerobic fermentation for 6 days. Based on the coupling of cell growth and D-mannitol production, metabolic evolution was used to improve D-mannitol production. After evolution for 80 generations, D-mannitol titer increased 2.6-fold and mannitol dehydrogenase activity increased 2.8-fold. Genetically stable strains constructed in this work could ferment sugars to produce D-mannitol without the addition of antibiotics, inducers and formate, which was favorable for industrial production.