Inorganic orthophosphate (Pi) is a primary form of phosphorus (P), one of the essential nutrients, acquired by the root and transported within plants. Long-distance communication between roots and shoots has to be achieved to sustain growth and ensure reproduction. Several molecules, such as Pi, sugars, hormones and microRNAs (miRNAs), have been recognized as systemic signals that convey the whole-plant Pi status internally. We have identified a systemic regulation by which plants control Pi transport to adapt to fluctuations in Pi availability, which involves the suppression of PHO2 encoding a ubiquitin conjugase by miR399. Upon Pi starvation, miR399 is upregulated first in shoots. It serves as a long-distance signal that moves through the phloem to roots, directing the cleavage of PHO2 transcripts and leading to enhanced Pi uptake and root-to-shoot translocation. To reveal the molecular mechanism underlying miR399 long-distance movement, we grafted with various mutants or transgenic lines expressing artificial miR399. We found that miR399 precursors are cell-autonomous and mature miR399 movement is independent of its biogenesis, sequence context, and length. Expressing viral silencing suppressor P19 in the root stele or blocking unloading in the root phloem pore pericycle (PPP) antagonized its silencing effect, suggesting that the miR399/miR399* duplex is a mobile entity unloaded through PPP. Notably, the scion miR399f level positively correlates with its amount translocated to rootstocks, implying dose-dependent movement. This study revealed how a Pi starvation-induced endogenous miRNA moves to coordinate shoot Pi demand with Pi acquisition and translocation activities in the root.