Prokaryotic organisms frequently use riboswitches to quantify intracellular metabolite concentration via high-affinity metabolite receptors. Riboswitches possess a metabolite-sensing system that controls gene regulation in a cis-acting fashion at the initiation of transcriptional/translational level by binding with a specific metabolite and controlling various biochemical pathways. Riboswitch binds with flavin mononucleotide (FMN), a phosphorylated form of riboflavin and controls gene expression involved in riboflavin biosynthesis and transport pathway. The first step of the riboflavin biosynthesis pathway is initiated by the conversion of guanine nucleotide triphosphate (GTP), which is an intermediate of the purine biosynthesis pathway. An alternative pentose phosphate pathway of riboflavin biosynthesis includes the enzymatic conversion of ribulose-5-phosphate into 3, 4 dihydroxy-2-butanone-4-phosphates by DHBP synthase. The product of ribAB interferes with both GTP cyclohydrolase II as well as DHBP synthase activities, which catalyze the cleavage of GTP and converts DHBP Ribu5P in the initial steps of both riboflavin biosynthesis branches. Riboswitches are located in the 5′ untranslated region (5′ UTR) of messenger RNAs and contain an aptamer domain (highly conserved in sequence) where metabolite binding leads to a conformational change in an aptamer domain, which modulate the regulation of gene expression located on bacterial mRNA. In this review, we focus on how riboswitch regulates the riboflavin biosynthesis pathway in Bacillus subtilis and Lactobacillus plantarum.