Lactococcus lactis has great potential for high-yield production of mannitol, which has not yet been fully realized. In this study, we characterize how the mannitol genes in L. lactis are organized and regulated, and use this information to establish efficient mannitol production. Although the organization of the mannitol genes in L. lactis was similar to that in other Gram-positives, mtlF and mtlD, encoding the Enzyme IIA component (EIIAmtl) of the mannitol phosphotransferase system (PTS), and the mannitol-1-phosphate dehydrogenase, respectively, were separated by a transcriptional terminator, and the mannitol genes were found to be organized in two transcriptional units: an operon comprising mtlA, encoding the Enzyme IIBC component (EIIBCmtl) of the mannitol PTS, mtlR, encoding a transcriptional activator, and mtlF, and a separately expressed mtlD. The promoters driving expression of the two transcriptional units were somewhat similar, and both contained predicted catabolite responsive elements (cre). Presence of carbon catabolite repression was demonstrated, and was shown to be relieved in stationary phase cells. The transcriptional activator MtlR (mtlR), in some Gram-positives, is repressed by phosphorylation by EIIAmtl, and when we knocked-out mtlF we indeed observed enhanced expression from the two promotors, which indicated that this mechanism was in place. Finally, by overexpressing the mtlD gene and using stationary phase cells as biocatalysts, we attained 10.1 g/L mannitol with a 55% yield, which is the highest titer ever reported for L. lactis. Summing up, the results of our study should be useful for improving the mannitol producing capacity of this important industrial organism. Importance Lactococcus lactis is the most studied species of the Lactic Acid Bacteria, and it is widely used in various food fermentations. To date, there have been several attempts to persuade L. lactis into producing mannitol, a sugar alcohol with important therapeutic and food applications. Until now, to achieve mannitol production in L. lactis, with significant titer and yield, it has been necessary to introduce and express foreign genes, which precludes the use of such strains in foods, due to their recombinant status. In this study, we systematically characterize how the mannitol genes in L. lactis are regulated, and demonstrate how this impacts on mannitol production capability. We harness this information and manage to establish efficient mannitol production, without introducing foreign genes.