Carbon metabolism is central to photosynthetic organisms and involves the coordinated operation and regulation of numerous proteins. In cyanobacteria, proteins involved in carbon metabolism are regulated by multiple regulators including the RNA polymerase sigma factor SigE, the histidine kinases Hik8, Hik31 and its plasmid-borne paralog Slr6041, and the response regulator Rre37. To understand the specificity and the cross-talk of such regulations, we simultaneously and quantitatively compared the proteomes of the gene knockout mutants for the regulators. A number of proteins showing differential expression in one or more mutants were identified, including four proteins that are unanimously upregulated or downregulated in all five mutants. These represent the important nodes of the intricate and elegant regulatory network for carbon metabolism. Moreover, serine phosphorylation of PII, a key signaling protein sensing and regulatingin vivocarbon/nitrogen (C/N) homeostasis through reversible phosphorylation, is massively increased with a concomitant significant decrease in glycogen content only in thehik8-knockout mutant, which also displays impaired dark viability. An unphosphorylatable PIIS49A substitution restored the glycogen content and rescued the dark viability of the mutant. Together, our study not only establishes the quantitative relationship between the targets and the corresponding regulators and elucidated their specificity and cross-talk but also unveils that Hik8 regulates glycogen accumulation through negative regulation of PIIphosphorylation, providing the first line of evidence that links the two-component system with PII-mediated signal transduction and implicates them in the regulation of carbon metabolism.