Cyanobacteria are the only prokaryotes that perform plant-like oxygenic photosynthesis. They evolved an inorganic carbon-concentrating mechanism to adapt to low CO2 conditions. Quantitative phospho-proteomics was applied to analyze regulatory features during the acclimation to low CO2 conditions in the model cyanobacterium Synechocystis sp. PCC 6803. Overall, more than 2500 proteins were quantified, equivalent to approximately 70% of the Synechocystis theoretical proteome. Proteins with changing abundances correlated largely with mRNA expression levels. Functional annotation of the non-correlating proteins revealed an enrichment of key metabolic processes fundamental for maintaining cellular homeostasis. Furthermore, 105 phospho-proteins harboring over 200 site-specific phosphorylation events were identified. Subunits of the bicarbonate transporter BCT1 and the redox switch protein CP12 were among phospho-proteins with significantly reduced phosphorylation levels at lower CO2, whereas the serine/threonine protein kinase SpkC revealed increased phosphorylation levels. The corresponding spkC mutant was characterized and showed decreased ability to acclimate to low CO2 conditions. Possible phosphorylation targets of SpkC including a BCT1 subunit were identified by phospho-proteomics. Collectively, our study highlights the importance of post-transcriptional regulation of protein abundances as well as post-translational regulation by protein phosphorylation for the successful acclimation towards low CO2 conditions in Synechocystis and possibly among cyanobacteria.