Organic carbon source plays a fundamental function in the metabolism and production of photosynthetic microorganisms. Herein, this study explored the effects of carbon source additions (glucose, sucrose and sodium acetate) on growth and biochemical components of the economic cyanobacterium Nostoc sphaeroides. The effects of sucrose and sodium acetate on its metabolomics changes, as well as the diversity and community composition of symbiotic bacteria, were analyzed. Results showed that sucrose noticeably enhanced the relative growth rate of N. sphaeroides. The content of phycocyanin was significantly greater under sodium acetate than under other carbon sources. Some metabolites associated with amino acid biosynthesis was significantly enriched under sodium acetate, implying its critical role in phycocyanin synthesis. Sodium acetate had a significant impact on the diversity and community composition of symbiotic bacteria. Additionally, partial symbiotic bacteria were shown to be involved in the amino acid metabolism of N. sphaeroides. These findings provided valuable insights into the mechanisms of N. sphaeroides on its metabolic responses to carbon source changes and the synergy of its symbiotic bacteria, contributing to the establishment of effective carbon source supply strategies for artificial cultivation and resource restoration.