Abstract Calcium ions (Ca2+) and Ca2+-related proteins mediate a wide array of downstream processes involved in plant responses to abiotic stresses. In Arabidopsis (Arabidopsis thaliana), disruption of the vacuolar Ca2+/H+ transporters CAX1 and CAX3 causes notable alterations in the shoot ionome, including phosphate (Pi) content. In this study, we showed that the cax1/cax3 double mutant displays an elevated Pi level in shoots as a result of increased Pi uptake in a miR399/PHO2-independent signaling pathway. Microarray analysis of the cax1/cax3 mutant suggests the regulatory function of CAX1 and CAX3 in suppressing the expression of a subset of shoot Pi starvation-responsive genes, including genes encoding the PHT1;4 Pi transporter and two SPX domain-containing proteins, SPX1 and SPX3. Moreover, although the expression of several PHT1 genes and PHT1;1/2/3 proteins is not up-regulated in the root of cax1/cax3, results from reciprocal grafting experiments indicate that the cax1/cax3 scion is responsible for high Pi accumulation in grafted plants and that the pht1;1 rootstock is sufficient to moderately repress such Pi accumulation. Based on these findings, we propose that CAX1 and CAX3 mediate a shoot-derived signal that modulates the activity of the root Pi transporter system, likely in part via posttranslational regulation of PHT1;1 Pi transporters.