Lignocellulosic materials, such as rice straw, are utilised as a substrate for microbial fermentation. Rice straw was pre-treated and used for fermentative production of D-lactic acid by genetic engineered Escherichia coli DX03 (ΔfrdBC ΔldhA ΔackA ΔpflB ΔpdhR::pflBp6-acEF-lpd ΔmgsA ΔadhE ΔptsG ΔmglB) in a 7 L fementer. After acid hydrolysis, detoxification and cellulase treatment, 52.4 g of reducing sugars was generated from 100 g of rice straw. Following a 36 h fermentation, 46.7 g of D-lactic acid was produced from the hydrolysate by DX03, achieving an optical purity of 99.8%, and a yield of 89.34%. This results suggests that E.coli strain DX03 has the potentials for fermentative production of green chemicals such as D-lactic acid, using the low-cost hydrolyzed lignocellulosic materials.