Cell–cell junctions are essential for multicellular organisms to maintain nutrient homoeostasis. A plant-type tight junction, the Casparian strip (CS)–Casparian strip membrane domain (CSD) that seals the paracellular space between adjacent endodermal cells, has been known for more than one hundred years. However, the molecular basis of this structure remains unknown. Here we report that a new family of proteins containing a glycine/alanine/proline-rich domain, a lectin domain and a secretory signal peptide (GAPLESS) mediates tethering of the plasma membrane to the CS in rice. The GAPLESS proteins are specifically localized in the CS of root endodermal cells, and loss of their functions results in a disabled cell–cell junction and disrupted nutrient homoeostasis. The GAPLESS protein forms a tight complex with OsCASP1 in the plasma membrane, thereby mediating the CS–CSD junction. This study provides valuable insights into the junctional complex of plant endodermal cells, shedding light on our understanding of nutrient homoeostasis in crops and the cell junctions of eukaryotes.