Suberin plays important roles in protecting plants against various stressors but the molecular mechanisms governing the regulated deposition of suberin are currently unclear. I provide evidence here that AtMYB53, AtMYB92, and AtMYB93 of the MYB-type transcription factor family are important regulators of suberin in root endodermis under non-stress conditions. I characterized an Arabidopsis steroid-inducible line of MYB53 showing significant suberin increase and a collection of loss-of-function mutants of MYB53/MYB92/MYB93 which exhibited major reductions of suberin in young roots, compared to wild-type. A suite of suberin biosynthetic genes were also positively regulated at the transcriptional level in MYB53 overexpression line and MYB53/MYB92/MYB93 loss-of-function lines.The identification of master regulators of suberin may provide the means to generate crops that are more stress resistant via enhancement of their suberized cell walls.