<p>Calcium (Ca²⁺) functions as a universal signaling ion across biological systems, yet its role in cyanobacterial metabolic regulation remains underexplored. Here, we investigated the association between Ca²⁺ availability and physiological responses in <i>Synechococcus elongatus</i> PCC 7942 in relation to the conserved regulatory protein PII. Ca²⁺ supplementation significantly increased cell viability, pigment accumulation, photosynthetic efficiency, and ATP/ADP ratios in wild-type cells, while a Δ<i>glnB</i> mutant lacking PII failed to respond, demonstrating PII’s central role. Notably, Ca²⁺ enhanced the stability of the trimeric form of PII, as demonstrated by size-exclusion chromatography, Native PAGE, and thermal aggregation assays. Computational modelling identified putative Ca²⁺-binding sites at subunit interfaces, suggesting that calcium coordination reinforces trimer integrity. Furthermore, Ca²⁺-stabilized PII supported carbon/nitrogen homeostasis by enhancing protein synthesis and reducing excess carbohydrate and lipid pools, while mitigating oxidative stress through reduced superoxide accumulation. These findings position PII as a key integrator linking calcium signaling to energy balance, photosynthetic performance, and stress resilience. Together, these findings uncover a previously unrecognized regulatory axis wherein Ca²⁺ enhances stability of PII, thereby coupling ionic signals to metabolic and physiological processes critical for cyanobacterial adaptation. Such regulatory networks represent promising targets for metabolic engineering strategies aimed at improving cyanobacterial growth and productivity in biotechnological applications.</p> Graphical abstract <p></p>