Protein phosphorylation is a principal signaling mechanism that mediates regulation of enzymatic activities, modulation of gene expression, and adaptation to environmental changes. Recent studies have shown a ubiquitous distribution of eukaryotic-type Serine/Threonine protein kinases in prokaryotic genomes, though the functions, substrates, and possible regulation of these enzymes remain largely unknown. In this study, we investigated whether cyanobacterial protein phosphorylation may be subject to redox regulation through modulation of the cysteine redox state, as has previously been reported for animals and plants. We also explored the role of a cyanobacterial Serine/Threonine kinase in oxidative stress tolerance.The Synechocystis sp. PCC 6803 Serine/Threonine kinase SpkB was found to be inhibited by oxidation and reactivated by thioredoxin-catalyzed reduction. A Synechocystis mutant devoid of the SpkB kinase was unable to phosphorylate the glycyl-tRNA synthetase β-subunit (GlyS), one of the most prominent phosphoproteins in the wild type, and recombinant purified SpkB could phosphorylate purified GlyS. In vivo characterization of the SpkB mutant showed a pronounced hypersensitivity to oxidative stress and displayed severe growth retardation or death in response to menadione, methyl viologen, and elevated light intensities.This study points out a previously unrecognised complexity of prokaryotic regulatory pathways in adaptation to the environment and extends the roles of bacterial eukaryotic-like Serine/Threonine kinases to oxidative stress response.The SpkB kinase is required for survival of the cyanobacterium Synechocystis sp. PCC 6803 under conditions implying increased concentrations of reactive oxygen species, and the activity of SpkB depends on the redox state of its cysteines.