Glycolysis is one of the best and widely conserved general metabolic pathways. Bacillus subtilis enzymes catalysing the central part of glycolysis, gathering the steps of interconversion of the triose phosphates from dihydroxyacetone-phosphate to phosphoenolpyruvate, are encoded by five genes, gapA, pgk, tpi, pgm and eno. They are transcribed in a hexacistronic operon together with cggR, the first cistron, encoding the repressor of this gapA operon. Using deletion analysis, we have localized the CggR operator between the promoter and the first gene of the operon. CggR was purified and used in gel mobility shift assays and DNase I footprinting experiments to delimit its target sequence. Site-directed mutagenesis and in vivo tests demonstrated that it consists of two direct-repeats (CGGGACN6TGTCN4CGGGACN6TG TC). Sequence analysis and transcriptome comparison of a wild-type and a cggR mutant strain strongly suggested that CggR regulates only the gapA operon. The presence of glycolytic carbon sources induces expression of the gapA operon. Genetic experiments allowed us to identify the metabolic steps required for the formation of the CggR effector. In vitro experiments with the suggested candidates allowed us to demonstrate that fructose-1,6-biphosphate (FBP) acts as an inhibitor of CggR DNA-binding activity (10 mM for full inhibition). FBP is thus the major signal for both CcpA-dependent catabolite repression (or activation) and activation of the central glycolytic genes. Genomic sequence comparisons suggest that these results can apply to numerous low-G+C, Gram-positive bacterial species.