Cyanobacteria are common components of the bacterioplankton in freshwater environments, where they play a key role as primary producers. Growth is limited by the availability of nutrients, particularly phosphate (Pi), and yet many species persist and flourish in environments with an unpredictable and constantly fluctuating supply of Pi. Genome analysis of the freshwater cyanobacterium Synechocystis sp. PCC6803 has identified that the membrane-bound transporter components of its Pho regulon consist of two high affinity (Pi) ABC transporters with multiple associated phosphate binding proteins (PBP), features in contrast to virtually all other known bacteria. Whilst the occurrence of duplicate ABC transporter mechanisms has been widely reported in freshwater cyanobacteria there are still very few reports that demonstrate the functional significance of individual, and apparently redundant, components of these ABC transporter systems. In previous work, disruption of one of the PBPs in Synechocystis sp. PCC6803, pstS1 (sll0680) led to an impairment in the expression of specific genes of the Pho regulon during Pi-deplete growth. This phenotype was not observed when the PBP from the second transporter was disrupted suggesting that each transporter could be functionally distinct. In this study 32Pi radiotracer uptake experiments using pst1 and pst2 deletion mutants showed Pst1 acts as a low affinity, high velocity transporter (Ks 3.7 ± 0.7 μM, Vmax 31.18 ± 3.96 fmol cell-1 min-1) and Pst2 a high affinity, low velocity system (Ks 0.07± 0.01 μM, Vmax 0.88 ± 0.11 fmol cell-1 min-1). Analysis of (qPCR) gene expression profiles and alkaline phosphatase activity also revealed how regulation of transporter abundance controls the nature of the Pi stress signal transduced by the SphS-SphR two component system. These Pi ABC transporters thus exhibit key differences in both their kinetic and regulatory properties, revealing a new strategy for the acquisition of phosphate that has potential implications for our understanding of the ecological success of this key microbial group.