Glucansucrases can perform acceptor reactions with different sugars to produce distinct oligosaccharides which can potentially act as prebiotics depending on their characteristics. Using cheap substrate sucrose and under mild and environmentally friendly conditions, glucansucrase-derived oligosaccharides can be produced with high yields. In this study, raffinose-derived oligosaccharides up to degree of polymerization (DP) 7 produced by glucansucrase E81 were characterized and their prebiotic functions were evaluated. The main product was a DP 4 oligosaccharide containing two products formed by the transfer of the glucose to raffinose at either the 3- (81%) or 4-O (19%) position of its galactose residue confirmed by NMR spectroscopy and LC-MS-LTQ analysis. The purified DP 4 raffinose-derived oligosaccharides were subjected to in vitro human gastric fluid and a-amylase digestion tests and no digestion was observed under both cases. Finally, prebiotic potential of DP 4 raffinose-derived oligosaccharides was tested with probiotic and pathogenic strains and only growth of Bifidobacteria promoted, thus supported the Bifidogenic activity of DP 4 raffinose-derived oligosaccharides.