Escherichia coli W, a sucrose-positive strain, was engineered for the homofermentative production of D-lactic acid through chromosomal deletion of the competing fermentative pathway genes (adhE, frdABCD, pta, pflB, aldA) and the repressor gene (cscR) of the sucrose operon, and metabolic evolution for improved anaerobic cell growth. The resulting strain, HBUT-D, efficiently fermented 100 g sucrose l(-1) into 85 g D-lactic acid l(-1) in 72-84 h in mineral salts medium with a volumetric productivity of similar to 1 g l(-1) h(-1), a product yield of 85 % and D-lactic acid optical purity of 98.3 %, and with a minor by-product of 4 g acetate l(-1). HBUT-D thus has great potential for production of D-lactic acid using an inexpensive substrate, such as sugar cane and/or beet molasses, which are primarily composed of sucrose.