Root development involves the maintenance of longitudinal cell division, cell expansion, and maturation, the systematic mechanisms that efficiently orchestrate root development remain elusive. Here, we identify a RALF-mediated intrinsic regulatory hub in rice that maintains meristem homeostasis and drives the transition from division to expansion. THESEUS3 (THE3), a CrRLK1L member homologous to THE1, interacts and phosphorylates RbohB to promote O2− generation in the RAM. OsRALF4 controls O2− homeostasis in root meristem through inhibiting THE3-RbohB signaling. O2− level, in turn, negatively regulates the stability of PMEs, suppressing pectin demethylesterification. With the decrease of O2−, demethylesterified pectin are generated and bound directly to OsRALF4, facilitating pectin condensation and cell elongation. To identify the RbohB interacting protein and PMEs induced by Tiron, we performed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.