The crhR gene for RNA helicase, CrhR, was one of the most highly induced genes when the cyanobacterium Synechocystis sp. PCC 6803 was exposed to a downward shift in ambient temperature. Although CrhR may be involved in the acclimatization of cyanobacterial cells to low-temperature environments, its functional role during the acclimatization is not known. In the present study, we mutated the crhR gene by replacement with a spectinomycin-resistance gene cassette. The resultant DeltacrhR mutant exhibited a phenotype of slow growth at low temperatures. DNA microarray analysis of the genome-wide expression of genes, and Northern and Western blotting analyses indicated that mutation of the crhR gene repressed the low-temperature-inducible expression of heat-shock genes groEL1 and groEL2, at the transcript and protein levels. The kinetics of the groESL co-transcript and the groEL2 transcript after addition of rifampicin suggested that CrhR stabilized these transcripts at an early phase, namely 5-60 min, during acclimatization to low temperatures, and enhanced the transcription of these genes at a later time, namely 3-5 h. Our results suggest that CrhR regulates the low-temperature-inducible expression of these heat-shock proteins, which, in turn, may be essential for acclimatization of Synechocystis cells to low temperatures.