Cyanobacterial carboxysomes encapsulate carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Genetic deletion of the major structural proteins encoded within the <
em>
ccm<
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operon in <
em>
Synechococcus<
/em>
sp. PCC 7002 (<
em>
?ccmKLMN<
/em>
) disrupts carboxysome formation and significantly affects cellular physiology. In this study, we employed both metabolite pool size analysis and isotopically nonstationary metabolic flux analysis (INST-MFA) to examine metabolic regulation in cells lacking carboxysomes. Under high CO<
sub>
2<
/sub>
environments (1%), the <
em>
?ccmKLMN<
/em>
mutant could recover growth and had a similar central flux distribution as the control strain, with the exceptions of moderately decreased photosynthesis and elevated biomass protein content and photorespiration activity. Metabolite analyses indicated that the <
em>
?ccmKLMN<
/em>
strain had significantly larger pool sizes of pyruvate (>
18 folds), UDPG (uridine diphosphate glucose), and aspartate as well as higher levels of secreted organic acids (e.g., malate and succinate). These results suggest that the <
em>
?ccmKLMN<
/em>
mutant is able to largely maintain a fluxome similar to the control strain by changing in intracellular metabolite concentrations and metabolite overflows under optimal growth conditions. When CO<
sub>
2<
/sub>
was insufficient (0.2%), provision of acetate moderately promoted mutant growth. Interestingly, the removal of microcompartments may loosen the flux network and promote RuBisCO side-reactions, facilitating redirection of central metabolites to competing pathways (i.e., pyruvate to heterologous lactate production). This study provides important insights into metabolic regulation via enzyme compartmentation and cyanobacterial compensatory responses.