Evidence suggests that heavy-metal tolerance can be induced in plants following pre-treatment with non-toxic metal concentrations, but the results are still controversial. In the present study, tobacco plants were exposed to increasing Zn²⁺ concentrations (up to 250 and/or 500 μM ZnSO₄) with or without a 1-week acclimation period with 30 μM ZnSO₄. Elevated Zn²⁺ was highly toxic for plants, and after 3 weeks of treatments there was a marked (≥50%) decline in plant growth in non-acclimated plants. Plant acclimation, on the other hand, increased plant dry mass and leaf area up to 1.6-fold compared with non-acclimated ones. In non-acclimated plants, the addition of 250 μM ZnSO₄ led to transient membrane depolarization and stomatal closure within 24h from the addition of the stress; by contrast, the acclimation process was associated with an improved stomatal regulation and a superior ability to maintain a negative root membrane potential, with values on average 37% more negative compared with non-acclimated plants. The different response at the plasma-membrane level between acclimated and non-acclimated plants was associated with an enhanced vacuolar Zn²⁺ sequestration and up to 2-fold higher expression of the tobacco orthologue of the Arabidopsis thaliana MTP1 gene. Thus, the acclimation process elicited specific detoxification mechanisms in roots that enhanced Zn²⁺ compartmentalization in vacuoles, thereby improving root membrane functionality and stomatal regulation in leaves following elevated Zn²⁺ stress.