In the complete annotated genome sequences of cyanobacterium Synechocystis sp. PCC 6803, one can find many putative genes for two-component response regulators that include a helix-turn-helix DNA-binding domain. The mRNA level of one of the putative genes, sll1330, was increased by glucose, especially in the presence of light. We successfully disrupted the sll1330 gene by targeted mutagenesis with a spectinomycin resistance cassette. Deltasll1330 could not grow well under light-activated heterotrophic growth conditions. Analyses of the expression of glycolytic genes revealed that the mRNA levels of five glycolytic genes, that is, glk (sll0593), pfkA (sll1196), fbaA (sll0018), gpmB (slr1124), and pk (sll0587), were decreased, and were regulated by Sll1330 under light and glucose-supplemented conditions. The Synechocystis sp. PCC 6803 genome each encodes two isozymes for these five glycolytic genes, suggesting that each of the two isozymes is regulated by Sll1330 at the mRNA level.