Because they are immotile organisms, higher plants have developed efficient strategies for adaptation to temperature changes. During cold acclimation, plants accumulate specific types of solutes to enhance freezing tolerance. The vacuole is a major solute storage organelle, but until now the role of tonoplast proteins in cold acclimation has not been investigated. In a comparative tonoplast proteome analysis, we identified several membrane proteins with altered abundance upon cold acclimation. We found an increased protein abundance of the tonoplast pyrophosphatase and subunits of the vacuolar V-ATPase and a significantly increased V-ATPase activity. This was accompanied by increased vacuolar concentrations of dicarbonic acids and soluble sugars. Consistently, the abundance of the tonoplast dicarbonic acid transporter was also higher in cold-acclimatized plants. However, no change in the protein abundance of tonoplast monosaccharide transporters was detectable. However, a generally higher cold-induced phosphorylation of members of this sugar transporter sub-group was observed. Our results indicate that cold-induced solute accumulation in the vacuole is mediated by increased acidification of this organelle. Thus solute transport activity is either modulated by increased protein amounts or by modification of proteins via phosphorylation.