Cell-to-cell communication is fundamental to multicellular life. In plants, plasmodesmata—cytoplasmic channels that connect adjacent cells—enable the transport of molecules between cells. In roots, such transport is thought to play a central role in nutrient acquisition and delivery across the multiple cell layers. In this study, we demonstrate that plasmodesmatal transport persists in fully differentiated Arabidopsis roots, even after the establishment of apoplastic barriers such as Casparian strips and suberin lamellae in the endodermis. This persistence highlights plasmodesmata as a critical pathway for intercellular trans-port in mature roots. We also identify a developmental switch in plasmodesmatal function: while transport is bidirectional in young roots, it becomes unidirectional toward the vasculature in differentiated roots. Through a genetic screen, we identified mutants with impaired directionality that maintain persistent bidirectional transport. These mutants show enlarged plasmodesmatal apertures due to defects in pectin composition and cell wall organization, highlighting the critical role of pectin in plasmodesmatal formation and function. Our findings reveal that plasmodesmata-mediated transport is dynamically regulated during root development and provide new insights into the cellular mechanisms underlying intercellular communication in plants.