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Cyanobacteria developed efficient carbon concentrating mechanisms which significantly improve the photosynthetic performance and survival of cells under limiting CO2conditions. Dynamic changes of the Synechocystisproteome to CO2limitation were investigated using shotgun LC−MS/MS approach with isobaric tag for relative and absolute quantification (iTRAQ) technique. Synechocystiscells grown at high (3%) CO2were shifted to air-level CO2followed by protein extraction after 6, 24, and 72 h. About 19% of the cyanobacterial proteome was identified and the expression changes were quantified for 17% of theoretical ORFs. For 76 proteins, up- or down-regulation was found to be significant (more than 1.5 or less than 0.7). Major changes were observed in proteins participating in inorganic carbon uptake, CO2fixation, nitrogen transport and assimilation, as well as in the protection of the photosynthetic machinery from excess of light. Further, a number of hypothetical proteins with unknown functions were discovered. In general, the cells appear to acclimate to low CO2without a significant stress since the stress-related molecular chaperones were down-regulated and only a minor decline was detected for proteins of phycobilisomes, photosynthetic complexes, and translation machinery. The results of iTRAQ experiment were validated by the Western blot analysis for selected proteins.