Heat shock proteins (Hsps) play a key role in the cellular defense response to diverse environmental stresses. Here, the role of Hsp genes in the acquisition of thermotolerance in the cyanobacterium Microcystis aeruginosa NIES-298 was investigated. Twelve Hsp-related genes were examined to observe their modulated expression patterns at different temperatures (10, 15, 25, and 35° C) over different exposure periods. HspA and HtpG transcripts showed an up-regulation of expression at low temperatures (10 and 15° C) and high temperature (35° C), compared with the control (25° C). To examine their effects upon thermotolerance, we purified recombinant HspA and HtpG proteins. During a thermotolerance study at 54° C, the HspA-transformed bacteria showed increased thermotolerance compared with the control. HtpG also played a role in the defense response to acute heat stress within 30 min. These findings provide a better understanding of cellular protection mechanisms against heat stress in cyanobacteria.