The sour taste of Citrus fruits is due to the extreme acidification of vacuoles in juice vesicle cells via a mechanism that remained elusive. Genetic analysis in petunia identified two vacuolar P-ATPases, PH1 and PH5, which determine flower color by hyperacidifying petal cell vacuoles. Here we show that Citrus homologs, CitPH1 and CitPH5, are expressed in sour lemon, orange, pummelo and rangpur lime fruits, while their expression is strongly reduced in sweet-tasting "acidless" varieties. Down-regulation of CitPH1 and CitPH5 is associated with mutations that disrupt expression of MYB, HLH and/or WRKY transcription factors homologous to those activating PH1 and PH5 in petunia. These findings address a long-standing enigma in cell biology and provide targets to engineer or select for taste in Citrus and other fruits.