Plant cells coated with hydrophobic compounds constitute a protective barrier to control movement of materials through plant tissues. In roots, the endodermis develops two barriers: the Casparian strips establish an apoplastic barrier and suberin lamellae prevent diffusion through the plasma membrane. Suberin is a complex biopolymer and its deposition is highly responsive to the environment. While the enzymatic framework involved in suberin biosynthesis is well characterized, subsequent steps in suberin formation and regulation remained elusive. Recent publications, studying suberin from a cell biological perspective, have enriched our knowledge on suberin transport and polymerization in the cell wall. These studies have also elucidated the molecular mechanisms controlling suberin biosynthesis and regulation as well as its physiological role in plant abiotic and biotic interactions.