The sulphidation of metal-based engineered nanoparticles (ENPs) presents a promising strategy to alleviate their ecotoxicity, particularly for ZnO ENPs used in plant growth enhancement. However, little is known about the interactions of sulphidized ENPs with plants, including their ionome. The key properties of ENPs that drive plant growth improvement can be significantly impacted by sulphidation. This study investigated the response of Zn-deficient barley to pristine (nZnO) and sulphidized ZnO ENPs (sulph-nZnO) at 0.5 mgZn L−1 under hydroponic conditions. The experimental approach evaluated plant biomass, elemental composition, and gene expression related to metal acquisition and homeostasis. Key findings revealed that Zn treatment of Zn-deficient plants showed higher Zn loading than the plants grown with the Zn source by 43–117%, and Zn distribution was primarily concentrated in shoots, in which the Zn level was as follows: nZnO < sulph-nZnO < ZnSO4. ENPs caused a comparable accumulation pattern of other metals (Fe, Mn, K, Ca) in barley shoots after 7 days, and their content was higher than ZnSO4 treatment. The transcript levels of most of the analyzed ZIP genes were similar regardless of the Zn compound treatments. In contrast, the gene expression related to vacuolar Zn sequestration and antioxidant mechanisms exhibited variability in the Zn-treated plants. In turn, the expression patterns of genes encoding Zn sequestration and antioxidant enzymes in barley shoots and roots did not directly correlate with total Zn content in plant tissues. However, the distinct transcriptional response may be associated with the ratios of different metals present. Although the spectroscopic and transcriptional profiles were generally consistent across ENP treatments, sulph-nZnO exhibited enhanced Zn uptake and elevated expression of ZIP1, a zinc-responsive gene involved in zinc efficiency. This suggests its potential as an innovative approach to improving plant elemental nutrition.