Updated publication reference for PubMed record(s): 29510212. Cyanobacteria fix atmospheric CO2 to biomass and through metabolic engineering can also act as photosynthetic cell factories for sustainable productions of fuels and chemicals. The Calvin cycle is the primary pathway for CO2 fixation in cyanobacteria, algae and C3 plants, and several studies have shown that overexpression of a cyanobacterial Calvin cycle enzyme, bifunctional sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (hereafter BiBPase), enhances CO2 fixation in both plants and algae, although its impact on cyanobacteria has not yet been rigorously studied. Here, we show that overexpression of BiBPase enhanced growth, cell size, and photosynthetic O2 evolution of the cyanobacterium Synechococcus sp. PCC 7002 in an environment with elevated CO2 concentration. Biochemical analysis, immunodetection, and proteomic analysis revealed that overexpression of BiBPase considerably elevated the cellular activities of two rate-limiting enzymes in the Calvin cycle, namely ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and aldolase, while it repressed several enzymes involved in the respiratory carbon metabolism (e.g. glycolysis and the oxidative pentose phosphate pathway) including glucose-6-phosphate dehydrogenase. Concomitantly, the content of glycogen was significantly reduced while the extracellular carbohydrate content increased. These results indicate that overexpression of BiBPase leads to global reprogramming of carbon metabolism in Synechococcus sp. PCC 7002, promoting photosynthetic carbon fixation and repressing the respiratory carbon catabolism, while altering carbohydrate partitioning.