Synechocystis sp. PCC 6803 PotD protein involved in polyamine transport was overexpressed in Escherichia coli as His-tagged PotD (rPotD). The purified rPotD showed saturable binding kinetics with radioactively-labeled polyamines. The rPotD exhibited a similar binding characteristic for three polyamines with a preference for spermidine. The Kd and maximum binding (Bmax) values for putrescine, spermidine and spermine were 13.2, 7.8 and 8.3 M, respectively and 0.74, 1.42 and 0.13 mol/mol rPotD, respectively. Optimum binding of rPotD with polyamines was at pH 8.0. Although the polyamine binding was increased by NaCl and sorbitol up to 10 mM, it was reduced when the concentration was higher than 50 mM. Competition experiments of polyamines analogs, such as amino acid, showed the specific binding of rPotD with polyamines. The in vivo function of Synechocystis PotD concerning polyamine transport was characterized by using Synechocystis knockout mutant disrupted potD gene (potD:Kmr). The mutant cells showed similar growth in BG11 to that of Synechocystis wild-type, although it was more sensitive to high concentration of putrescine or spermidine up to 0.5 mM than wild-type cells. Growth of Synechocystis cells in the medium containing either 0.5 mM putrescine or spermidine led to an increase in PotD levels, about 1.6 and 2.8 fold, respectively. The presence of 550 mM NaCl in BG11 did not significantly affect the Synechocystis growth in both wild-type and mutant cells, while the presence of 300 mM sorbitol in BG11 resulted in the growth stimulation in wild-type and growth inhibition in mutant cells. The putrescine and spermidine uptakes were induced by either NaCl or sorbitol up to 10 mM. Interestingly, the reduction of spermidine uptake activity was observed by 50% in mutant cells, while putrescine uptake activity was not altered. Additionally, the spermidine excretion activity was decreased by 60% in mutant cells. High light showed an inhibitory effect on Synechocystis growth in both wild-type and mutant cells. These mutant cells were showed growth inhibition after the first two days. Besides, the high light enhanced the putrescine and spermidine uptake activity by 3 and 2.5 fold, respectively. Furthermore, the external polyamines stimulated both PSII activity and photosynthetic capacity within 30 min but decreased sequentially after one hour in both wild-type and mutant cells. Docking of these polyamines into the homology model of Synechocystis PotD showed that all three polyamines are able to interact with Synechocystis PotD. The binding modes of the docked putrescine and spermidine in Synechocystis are similar to those of PotF and PotD in E. coli, respectively. The overall results support the role of PotD in mediating polyamine transport in Synechocystis sp. PCC 6803.