Understanding the interaction of macro- and micronutrients is a prerequisite to targeting nutrient balance in crop production. A 3-year field study was conducted to determine mineral nutrient uptake of maize hybrids with N fertilizer application under different rotation systems. The experiment was arranged in a split-plot design with rotation [maize-alfalfa (MA), maize-soybean (MS), and continuous maize (MM)] by N rate (0, 50, 100 and 150 kg N ha(-1)) as the mainplot and hybrid as the subplot. Two additional treatments (200 and 250 kg N ha(-1)) were tested in MM. Maize plant total Mg, Zn, and Cu content were in the order: MA MS MM. Plant Fe uptake was the lowest in MA and not affected by N input. The increased Cu uptake with increasing N rates indicated the synergism of these two nutrients, whereas dilution effects of N application on stover Zn and Mg concentrations were recorded. Rotation systems and N rates interactively affected nutrient harvest index and internal efficiency of Zn, Mg, Fe, and Cu. Relationships of plant N with Cu and Mg concentrations, and N with Zn, Mg, and Cu content at the V6 stage were established, but they were not necessarily preserved at maturity due to the progressive synergistic and dilution effects. The findings of nutrient uptake of Cu, Zn, Mg and Fe and their relationships with N nutrition in maize with stacked transgenic traits are important for developing best management practices to achieve concurrent improvements in nutrient use efficiency and crop productivity.