Glycosyltransferases of lactic acid bacteria are associated with biofilm formation, bacterial stress response and sucrose metabolism. The aim of this study was to determine the contribution of glycosyltransferases to sucrose metabolism in Lactobacillus reuteri TMW1.106 expressing the glucosyltransferase GtfA and the inulosucrase Inu, and L. reuteri LTH 5448 expressing the fructosyltransferase FtfA. Transcriptional analysis using quantitative real time PCR revealed that expression of ftfA of L. reuteri LTH5448 was induced by sucrose, while sucrose had no effect on gtfA and inu expression of strain TMW 1.106. Inactivation of ftfA had no influence on growth of L. reuteri LTH5448 and only a minor impact on sucrose turnover. L. reuteri TMW1.106 and its gtfA and inu mutants reached similar cell counts when maltose was offered as substrate. Mutation of gtfA or inu impaired growth in media containing sucrose as sole carbon source despite the expression of sucrose phosphorylase as an alternative sucrose-hydrolysing enzyme. Moreover, the gtfA and inu mutants formed less lactate and ethanol and tolerated lower lactate levels compared to L. reuteri TMW1.106. The inu mutant constitutively overexpressed GtfA. We show here that the impact of different glycosyltransferases on sucrose metabolism of L. reuteri is strain dependent. In strain L. reuteri TMW 1.106, GtfA accounts for sucrose utilization, metabolism, and growth of the organism. In contrast, FtfA of L. reuteri LTH5448 contributes to sucrose turnover but alternative routes for sucrose metabolism are functional in this strain. Our data thus indicate that these glycosyltransferases affect the competitiveness of some L. reuteri strains in ecosystems where sucrose is present.