Glycosyltransferases (GTs) are enzymes that play important roles in various biological processes, such as photosynthesis, cell motility, exopolysaccharides, and lipid metabolism, by transferring sugars to different targets. However, the regulation of carbon metabolism by GTs in Synechocystis has not been reported yet. To identify novel GTs proteins involved in carbon metabolism, we performed a large-scale screening of GT gene-deletion mutants in Synechocystis. Our results revealed that the glycosyltransferase Slr1064, which is mainly distributed in the membrane compartment, is essential for the growth of Synechocystis under heterotrophic and mixotrophic conditions (HT and MT), but not under autotrophic conditions (AT). Integrated proteomic and metabolic analyses showed that Slr1064 regulates the Calvin cycle by modulating the levels of UDP-GlcNAc, which have been reported to mediate O-GlcNAc modification of Gap2. Further analyses demonstrated that the Δslr1064 mutant has a lower turnover rate of Gap2 under MT condition and shows dissociation of the PRK/GAPDH/CP12 complex under dark culture conditions. These findings provide new insights into the role of GTs in carbon metabolism in Synechocystis and expand our understanding of the regulatory mechanism of carbon metabolism in this important model organism.