Rho guanosine triphosphatases (GTPases) are master regulators of cell signaling, however they’re regulated depending upon the cellular context is uncertain. We found the phospholipid phosphatidylserine functions as a developmentally controlled lipid rheostat that tunes Rho GTPase signaling in Arabidopsis. Live superresolution single-molecule imaging demonstrated that the protein Rho of Plants 6 (ROP6) is stabilized by phosphatidylserine into plasma membrane nanodomains, which are required for auxin signaling. Our experiments also revealed that the level of phosphatidylserine modulates the number of ROP6 nanoclusters induced by auxin and hence signaling, including regulation of gravitropism and endocytosis and that the plasma membrane phosphatidylserine content changes during plant root growth. Our work proves that variations in amounts are a process which may be leveraged to modulate GTPase.