The phosphorylation states of two serine residues within the C-terminal domain of AtPIP2;1 (S280, S283) regulate its trafficking to the plasma membrane in response to salt and osmotic stress. Here we investigated whether the phosphorylation states of S280 and S283 also influence AtPIP2;1 facilitated water and cation transport. A series of single and double S280 and S283 phospho-mimic and -deficient AtPIP2;1 mutants were tested in heterologous systems. In Xenopus laevis oocytes, phospho-mimic mutants AtPIP2;1 S280D, S283D and S280D/S283D, had significantly greater ion conductance for Na+ and K+, whereas the S280A single and S280A/S283A double mutants, had greater water permeability. A phospho-mimic-dependent inverse relationship between AtPIP2;1 water and ion transport with a 10-fold change in both was observed. These results revealed that phosphorylation of S280 and S283 influences the preferential facilitation between ion and water permeability by AtPIP2;1. The results also hint at other sites playing a role that are yet to be elucidated. Expression of the phospho-mimic AtPIP2;1 mutants in Saccharomyces cerevisiae, confirmed that phosphorylation influences plasma membrane localisation, and revealed higher Na+ accumulation for S280A and S283D. Collectively, the results show that phosphorylation in the C-terminal domain of AtPIP2;1 influences its subcellular localisation and cation transport capacity.