Previous RNA sequencing has allowed identifying 129 long 5'-UTRs in the L. lactis MG1363 transcriptome. These sequences potentially harbor cis-acting riboswitches. One of the identified extended 5'-UTRs is a putative thiamine pyrophosphate (TPP) riboswitch. It is located immediately upstream of the thiamine transporter gene thiT (llmg_0334). To confirm this assumption, the 5'-UTR sequence was placed upstream of the gene encoding the super folder green fluorescent protein, sfgfp, allowing examining the expression of sfGFP in the presence or absence of thiamine in the medium. The results show that this sequence indeed represents a thiamine-responsive TPP riboswitch. This RNA-based genetic control device was used to successfully restore the mutant phenotype of an L. lactis strain lacking the major autolysin gene acmA. The L. lactis thiT TPP riboswitch (RSthiT) is a useful molecular genetic tool enabling to gradually downregulate the expression of genes under its control by adjusting thiamine concentration. Importance The capacity of microbes with biotechnological importance to adapt and survive under quickly changing industrial conditions depends on their ability to adequately control gene expression. Riboswitches are important RNA-based elements involved in rapid and precise gene regulation. Here we present the identification of a natural thiamine-responsive riboswitch of Lactococcus lactis, a bacterium used worldwide in the production of dairy products. We used it to restore a genetic defect in an L. lactis mutant and show that it is a valuable addition to the ever-expanding L. lactis genetic toolbox.