Arsenate (As(V)) is an analog of phosphate (Pi), and previous studies have shown that As(V) and Pi are absorbed via the same transport systems in some organisms. However, little is known about which periplasmic phosphate-binding proteins (PBPs) of ABC-type Pi transporters play major roles in As(V) uptake by cyanobacteria. In this study, the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) was chosen to study the ability of PBPs to discriminate between Pi and As(V). We found that As(V) and Pi competed for uptake via Pi transporters when Synechocystis was treated with As(V) and/or Pi in short-term incubation. The As/P molar ratios of Synechocystis wild type (WT) and mutants (∆sphX, ∆pstS2, ∆sll0540, and ∆sphXpstS1) were measured, and they were significantly different with the order WT > ∆pstS2 > ∆sll0540 > ∆sphXpstS1 > ∆sphX. Furthermore, As(V) uptake by Escherichia coli strain Transetta expressing PBP genes, particularly sphX or sll0540, was significantly increased when PBP gene (pstS1, pstS2, sphX, or sll0540) was separately expressed in Transetta at the same level. Subsequent phylogenetic analyses of PBPs showed that SphX and Sll0540 belonged to the low-affinity PBPs, while PstS1 and PstS2 were clustered with the high-affinity PBPs. These results implied that As(V) was taken up via Pi transporters, and the low-affinity PBPs, SphX and Sll0540, made a significant contribution to As(V) uptake.