Casparian strips (CSs) are cell wall modifications of vascular plants restricting extracellular free diffusion into and out of the vascular system [1]. This barrier plays a critical role in controlling the acquisition of nutrients and water necessary for normal plant development [2, 3, 4, 5]. CSs are formed by the precise deposition of a band of lignin approximately 2 μm wide and 150 nm thick spanning the apoplastic space between adjacent endodermal cells [6, 7]. Here, we identified a copper-containing protein, Uclacyanin1 (UCC1), that is sub-compartmentalized within the CS. UCC1 forms a central CS nanodomain in comparison with other CS-located proteins that are found to be mainly accumulated at the periphery of the CS. We found that loss-of-function of two uclacyanins (UCC1 and UCC2) reduces lignification specifically in this central CS nanodomain, revealing a nano-compartmentalized machinery for lignin polymerization. This loss of lignification leads to increased endodermal permeability and, consequently, to a loss of mineral nutrient homeostasis.