Background <p>Plants are continuously exposed to diverse environmental stresses that impair growth and development. Sensing environmental cues and converting them into intracellular signals is crucial in mounting an appropriate stress response. Calcium (Ca²⁺) is a key secondary messenger that oscillates in response to specific environmental stimuli. The transient change in Ca²⁺ levels is decoded by Ca²⁺ sensors to elicit downstream stress responses. Here, we report the regulation of a tonoplast intrinsic protein (TIP2;1) expression through calcium signaling in <i>Withania somnifera</i> under osmotic stress.</p> Methods and results <p>In this study, osmotic stress triggers a transient spike in total Ca²⁺ content at 60&#xa0;min, followed by a gradual decline. This change was accompanied by strong upregulation of TIP2;1 (16-fold) and calcium-dependent protein kinase 11 (CDPK11, 34-fold). EGTA-mediated chelation of Ca²⁺ markedly suppressed TIP2;1 but enhanced CDPK11 expression. In contrast, supplementation with exogenous Ca²⁺ restored TIP2;1 expression. Stress-induced stomatal closure and maintenance of Na⁺/K⁺ ratio in shoots were further correlated with upregulation of the potassium transporter 3-like (POT) transcript. Osmotic stress also caused a significant decline in chlorophyll content, indicating compromised photosynthetic efficiency. Activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) were strongly increased to mitigate oxidative stress.</p> Conclusion <p>Our findings revealed that PEG-induced osmotic stress triggers TIP2;1 expression in a calcium-dependent manner. Calcium signaling orchestrates the balance of Na⁺/K⁺ ratio, chlorophyll stability, stomatal dynamics, and antioxidant defense. A precise understanding of these mechanisms could help in developing strategies to minimize the adverse effects of drought on plant productivity.</p>