Dextrans with distinct molecular size and structure are increasingly being used in the food and pharmaceutical industries. Dextran is produced by dextransucrase (DSR, EC2.4.5.1), which is produced by Leuconostoc mesenteroides. DSR belongs to glycosyl hydrolase family (GH70) and synthesizes branched alpha-glucan (dextran) with both 5% alpha(1-3) and 95% alpha(1-6) glycosidic linkages. The DSR gene dex-YG (Genebank, Accession No. DQ345760) was cloned from the wild strain Leuconostoc mesenteroides 0326. This study generated a series of C-terMinally truncated variants of dextransucrase and substituting the amino-acid residues in the active site of DSR. With shorter length of DSR, its polysaccharide-synthesizing capability was impaired heavily, whereas oligosaccharide (acting as prebiotics)-synthesizing capability increased significantly, efficiently producing special sizes of dextran. All truncated mutant enzymes were active. Results demonstrated that the catalytic domain dextransucrase was likely in 800 as or less. Based on the three-dimensional structure model of dextransucrase built through homology modeling methods, the DSR and its mutants with the acceptor substrate of maltose and donor substrate of sucrose were studied by molecular-docking method. Substituting these amino acid residues significantly affected enzyme activities. Compared with the wild-type dextran, mutant enzymes catalyzed the synthesis of alpha-glucan with 1-9% alpha(1-3) and 90-98% alpha(1-6) branching linkages. Some mutants introduced a small amount of alpha(1-4) linkages and alpha(1-2) linkages. This strategy can be effectively used for the rational protein design of dextransucrase.