Nitric oxide synthases (NOSs) are heme-based monooxygenases that catalyze the NADPH-dependent oxidation of L-arginine to produce NO and L-citrulline. Over the past five years, the identification and characterization of NOS homologs in cyanobacteria have significantly advanced our understanding of these enzymes. However, the precise mechanisms through which NOS-derived NO influences nitrogen metabolism remain incompletely elucidated. Therefore, the present study aims to investigates the impact of NOS-derived NO on nitrogen metabolism, heterocyte development, and carbon utilization dynamics in Aphanizomenon flos-aquae. Results demonstrate a three-fold increase in NOS-dependent NO production during the log to stationary growth phase in reponse to L-arginine availability. This increase in NOS activity substantially impacted critical cellular processes related to nitrogen metabolism. Specifically, the inhibition of NOS activity disrupted regulatory mechanisms involving ntcA and glnB genes, resulting in a failure to induce hetR, hep, dev and nif genes necessary for heterocyte differentiation and nitrogenase synthesis. NOS-derived NO also played a pivotal role in modulating the glutamine synthetase-glutamate synthase (GS-GOGAT) pathway, as evidenced by the sharp decline in glutamine and glutamate levels under NOS inhibition, which indicates impaired nitrogen assimilation. Besides, the observed alterations in succinate, fumarate, malate and pyruvate suggest regulatory roles of NOS in energy metabolism. NOS-inhibited cells redirected carbon flux towards glycogen/lipid biosynthesis, alongside protein degradation causing chlorosis, indicating nitrogen deficiency and compromised cellular viability. In contrast, NOS elicitation enhanced metabolic activity, supporting nitrogen assimilation and cellular growth. Overall, our results revealed the complex relationship among NOS-derived NO signaling, nitrogen metabolism, and carbon flux in cyanobacteria.