Key message <p>Calcium plays a role in modulating aquaporin expression, reducing oxidative stress, preserving chlorophyll content, and maintaining membrane integrity, suggesting it may enhance eucalypt resilience to water restriction.</p> Abstract <p>Drought is a significant abiotic stress that severely impacts the growth and productivity of forests worldwide. This study investigates the role of calcium (Ca²⁺) supply in modulating aquaporin (AQP) gene expression and mitigating oxidative stress in eucalypt (<i>Eucalyptus urophylla</i> × <i>Eucalyptus camaldulensis</i>) under water restriction. Three-month-old eucalypt seedlings were subjected to water restriction, with or without Ca²⁺ supplementation. We measured mRNA expression levels of five aquaporin isoforms (<i>PIP1;2, PIP1;3, PIP2;1, PIP2;2,</i> and <i>PIP2;7</i>), chlorophyll content, and markers of oxidative stress [e.g., hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and the activities of catalase (CAT) and superoxide dismutase (SOD)]. Our findings reveal that initial water restriction upregulated the expression of <i>PIP1;2, PIP2;1, PIP2;2,</i> and <i>PIP2;7</i> in roots, which likely facilitated water uptake and maintained cellular hydration. However, under prolonged water limitation, plants supplemented with Ca²⁺ showed downregulation of these PIPs, possibly as a protective mechanism to reduce water loss. In contrast, plants without Ca²⁺ exhibited an upregulation of <i>PIP1;2, PIP1;3</i>, and <i>PIP2;7</i> under prolonged water deficit. Ca²⁺ supplementation also played a crucial role in preserving chlorophyll content and reducing oxidative damage under water limitation. Plants supplemented with Ca²⁺ had lower levels of H₂O₂ and MDA, reduced oxidative damage and membrane permeability, alongside increased CAT activity, which suggests an enhanced oxidative response. Our results provide new insights into how Ca²⁺ enhances eucalypt responses to water deprivation, enhancing our understanding of the function of Ca²⁺ in drought responses.</p>