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Calcium-dependent protein kinases (CDPK) are implicated in signaling transduction in eukaryotic organisms. It is largely unknown whether <i>StCDPK28</i> plays a role in the response to water deficiency and osmotic stress in potato plants (<i>Solanum tuberosum</i> L.). Potato cv. Zihuabai was cultivated under natural, moderate, and severe water deficiency conditions; to induce osmotic stress, potato plants were treated with 10% or 20% PEG. <i>StCDPK28</i>-overexpression and <i>StCDPK28</i>-knockdown plants were constructed. <i>StCDPKs</i> were evaluated by qRT-PCR. The subcellular location of the StCDPK28 protein was observed with confocal scanning laser microscopy. Phenotypic changes were indicated by photosynthetic activity, the contents of H₂O₂, MDA and proline, and the activities of CAT, SOD and POD. Results showed water deficiency and osmotic stress altered <i>StCDPK</i> expression patterns. StCDPK28 exhibited a membrane, cytosolic and nuclear localization. Water deficiency and osmotic stress induced <i>StCDPK28</i> upregulation. Photosynthetic activity was enhanced by <i>StCDPK28</i> overexpression, while decreased by <i>StCDPK2</i> knockdown under water deficiency and osmotic stress. <i>StCDPK28</i> overexpression decreased H₂O₂ and MDA, and increased proline, while <i>StCDPK28</i> knockdown showed reverse results, compared with the wild type, in response to water deficiency and osmotic stress. <i>StCDPK28</i> overexpression increased the activities of CAT, SOD and POD, while <i>StCDPK28</i>-knockdown plants indicated the reverse trend under water deficiency and osmotic stress conditions. Regulation of StCDPK28 expression could be a promising approach to improve the tolerance ability of potato plants in response to drought or high salt media.