Aquaporins form a family of water and solute channel proteins and are present in most living organisms. In plants, aquaporins play an important role in the regulation of root water transport in response to abiotic stresses. In this work, we investigated the role of phosphorylation of Plasma membrane Intrinsic Protein (PIP) aquaporins in the Arabidopsis thaliana root by a combination of quantitative mass spectrometry and cellular biology approaches. A novel phosphoproteomic procedure that involves plasma membrane purification, phosphopeptide enrichment with TiO2 columns, and systematic mass spectrometry sequencing revealed multiple and adjacent phosphorylation sites in the C-terminal tail of several AtPIPs. Six of these sites had not been previously described. The phosphorylation of AtPIP2;1 at two C-terminal sites (Ser280, Ser283) was monitored by an Absolute Quantification method and shown to be altered in response to treatments of plants by salt (NaCl) and hydrogen peroxide. The two treatments are known to strongly decrease the water permeability of Arabidopsis roots. To investigate a putative role of Ser280 and Ser283 phosphorylation in aquaporin sub-cellular trafficking, AtPIP2;1 forms mutated at either one of the two sites were fused to the Green Fluorescent Protein and expressed in transgenic plants. Confocal microscopy analysis of these plants revealed that, in resting conditions, phosphorylation of Ser283 is necessary to target AtPIP2;1 to the plasma membrane. In addition, a NaCl treatment induced an intracellular accumulation of AtPIP2;1 by exerting specific actions onto AtPIP2;1 forms differing in their phosphorylation at Ser283 to induce their accumulation in distinct intracellular structures. Thus, the present study documents stress-induced quantitative changes in aquaporin phosphorylation and establishes for the first time a link with aquaporin sub-cellular localization.