Although nitrogen (N) is known to affect mineral element homeostasis in plants, the molecular mechanisms of interactions between N and other nutrients remain largely unclear. We report here that N supply affects ion homeostasis in maize. Berberine hemisulfate staining and a propidium iodide penetration assay showed that N luxury significantly delayed Casparian strip (CS) formation in maize roots. We further demonstrated that N-mediated CS formation in maize was independent of RBOHF-activated HO production. N luxury induced the expression of ZmmiR528 in whole roots and root tips. Knockdown and loss-of-function of ZmmiR528 promoted CS formation under both N-luxury and N-deficient conditions. Both ZmMIR528a and ZmMIR528b contribute to early CS formation under different N conditions. RNA-seq and real-time RT-PCR analysis demonstrated that ZmLAC3, but not ZmLAC5, responded to N treatments. Consistent with results obtained with ZmmiR528 TM transgenic maize and mir528a/b loss-of-function mutants, transgenic maize overexpressing ZmLAC3 displayed early CS formation under different N conditions. Under field conditions, K, Ca, Mn, Cu, Mg, and Zn concentrations were greater in the ear leaf of ZmLAC3-overexpressing transgenic maize than in the wild type. These results indicate that ZmmiR528 affects CS formation in maize by regulating the expression of ZmLAC3, and modification of CS formation has the potential to improve maize quality.