Significance Endodermal suberin plays crucial roles in plant nutrition by forming barriers for the free diffusion of water and nutrients. Suberin formation is tightly regulated by exogenous and developmental cues, which independently control suberin deposition in the endodermis. A set of four MYB transcription factors, expressed in the endodermis and responding to suberin-inducing conditions, individually prompt suberization and include the MYBs required for suberin formation and regulation. Overexpressing these transcription factors specifically in the endodermis or mutating all four led to plants specifically overproducing or underproducing endodermal suberin, respectively. Physiological analysis of these plants allowed us to decipher the role of suberin independently of synthetic transgenes and other endodermal barriers.