The regulatory mechanisms underlying ploidy dynamics in cyanobacteria under phosphorus (P) limitation remain poorly understood. In this study, we investigated the impact of phosphate deprivation on the polyploidy of Synechocystis sp. PCC 6803 through integrated approaches combining spectrofluorometry, flow cytometry, comparative transcriptomics, Pho regulon prediction, and enzymatic activity assays. Our results revealed a significant reduction in genomic DNA content (P < 0.01) during cultivation in phosphate-free BG11 medium, with average genome copy numbers decreasing from 24.34 +/- 0.27 in standard BG11 to 6.18 +/- 0.25 under P-depleted conditions (P < 0.01). Transcriptomic analysis demonstrated marked upregulation of genes associated with two-component signaling systems, ABC transporters, and nucleotide metabolism, while DNA replication, homologous recombination, and mismatch repair pathways were significantly downregulated (P < 0.05). Concurrently, alkaline phosphatase activity exhibited a substantial increase (P < 0.01), suggesting enhanced phosphate mobilization. These findings collectively indicated that genome copy number in Synechocystis sp. PCC 6803 was dynamically regulated through the coordinated interplay between DNA replication suppression and degradation activation in response to phosphorus availability. This work provides novel insights into the molecular basis of ploidy regulation in cyanobacteria and offers valuable implications for understanding analogous mechanisms in chloroplasts.