Simultaneous catabolic and anabolic glucose metabolism occurs in the same compartment during photomixotrophic growth of the model cyanobacterium Synechocystis sp. PCC 6803. The presence of glucose is stressful to the cells; it is reflected in the high frequency of suppression mutations in glucose-sensitive mutants. We show that glucose affects many cellular processes. It stimulates respiration and the rate of photosynthesis and quantum yield in low- but not high-CO2-grown cells. Fluorescence and thermoluminescence parameters of photosystem II are also affected but the results did not lend support to sustained glucose driven over reduction in the light. Glucose-sensitive mutants such as ΔpmgA (impaired in photomixotrophic growth) and Δhik31 (lacking histidine kinase 31) are far more susceptible under high than low air level of CO2. A glycine to tryptophan mutation in position 354 in NdhF3, involved in the high-affinity CO2 uptake, rescued ΔpmgA. A rise in the apparent photosynthetic affinity to external inorganic carbon is observed in high-CO2-grown wild-type cells after the addition of glucose, but not in mutant ΔpmgA. This is attributed to upregulation of certain low-CO2-induced genes, involved in inorganic carbon uptake, in the wild type but not in ΔpmgA. These data uncovered a new level of interaction between CO2 fixation (and the CO2-concentrating mechanism) and photomixotrophic growth in cyanobacteria.