alpha-Galacto-oligosaccharides (alpha-GOS) are produced by transgalactosylation reactions of alpha-galactosidase (alpha-Gal) or by conversion of raffinose family oligosaccharides by levansucrase. Similarly to beta-GOS, alpha-GOS have the potential to mimic glycan receptors on eukaryotic cells and act as molecular decoys to prevent bacterial infection; however, data on transgalactosylation reactions of alpha-Gal remain scarce. The alpha-Gal gene sequence from Lactobacillus reuteri was cloned into an alpha-Gal negative strain of Lactococcus lactis. Transgalactosylation reactions were achieved using crude cell extracts with melibiose or raffinose as galactosyl donor and fucose, N-acetylglucosamine or lactose as galactosyl acceptor. The composition, sequence and most linkage types of alpha-GOS formed with acceptors saccharides were determined by liquid chromatography-tandem mass spectrometry. alpha-Gal of Lactobacillus reuteri formed (1 -> aEuro parts per thousand 3)-, (1 -> aEuro parts per thousand 4)- or (1 -> aEuro parts per thousand 6)-linked alpha-GOS but exhibited a preference for formation of (1 -> aEuro parts per thousand 6)-linkages. Fucose, N-acetylglucosamine and lactose were suitable galactosyl acceptors for alpha-Gal of L. reuteri, resulting in formation of (1 -> aEuro parts per thousand 3)-, (1 -> aEuro parts per thousand 4)- or (1 -> aEuro parts per thousand 6)-linked hetero-oligosaccharides. By determining the structural specificity of alpha-Gal and increasing the variation of oligosaccharides produced by introducing alternative acceptor sugars, this work supports further studies to assess alpha-GOS pathogen adhesion prevention in mammalian hosts.