Abstract: Isolation and identification of acid-producing bacteria from environmental samples is still one of important and effective approaches to obtain strains producing lactic acid with high optical purity. In this study, an isolate strain named HL64-1, producing D-lactic acid with high optical purity, was isolated from rotten fruit. And HL64-1 was characterized as Leuconostoc pseudomesenteroides based on morphology, 16S rDNA sequence analysis and sequence similarity Blast analysis. After fermentation for 24 h, 62.18 g/L D-lactic acid was produced with an optical purity of 99.90%(ee) in basic fermentation culture. The acid production rate was 2.59 g/(L·h). Scale-up fermentation of L. pseudomesenteroides HL64-1 in 5-L fermenter for 72 h produced 78.74 g/L D-lactic acid by replenishment of carbon sources, and the average acid production rate was 1.09 g/(L·h). Moreover, L. pseudomesenteroides HL64-1 was found to be capable of using effectively cheap raw materials, such as agricultural by-products peanut cake meal and cottonseed meal, as alternative nitrogen sources, leading a reduction of fermentation cost. In addition, L. pseudomesenteroides HL64-1 could metabolize xylose into D-lactic acid. Co-fermentation of glucose and xylose mixtures showed that glucose significantly enhanced the utilization efficiency of xylose, making L. pseudomesenteroides HL64-1 to be a promising strain for pure D-lactic acid industrial production.