The Golgi-located phosphate exporter PHT4;6 has been described as involved in salt tolerance but further analysis on the physiological impact of PHT4;6 remained elusive. Here we show that PHT4;6–GFP is targeted to thetrans-Golgi compartment and that loss of function of this carrier protein has a dramatic impact on plant growth and development. Knockout mutants ofpht4;6exhibit a dwarf phenotype that is complemented by the homologous gene from rice (Oryza sativa). Interestingly,pht4;6mutants show altered characteristics of several Golgi-related functions, such as an altered abundance of certain N-glycosylated proteins, altered composition of cell-wall hemicelluose, and higher sensitivity to the Golgi α-mannosidase and the retrograde transport inhibitors kifunensine and brefeldin A, respectively. Moreover,pht4;6mutants exhibit a ‘mimic disease’ phenotype accompanied by constitutively activated pathogen defense mechanisms and increased resistance against the virulentPseudomonas syringaestrain DC3000. Surprisingly,pht4;6mutants also exhibit phosphate starvation symptoms, as revealed at the morphological and molecular level, although total Pi levels in wild-type andpht4;6plants are similar. This suggested that subcellular Pi compartmentation was impaired. By use of nuclear magnetic resonance (NMR), increased Pi concentration was detected in acidic compartments ofpht4;6mutants. We propose that impaired Pi efflux from thetrans-Golgi lumen results in accumulation of inorganic phosphate in other internal compartments, leading to low cytoplasmic phosphate levels with detrimental effects on plant performance.