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With persistent elevation in global temperature, water scarcity becomes a major threat to plant growth and development, yield security, agricultural sustainability, and food production. Proline, as a key osmolyte and antioxidant, plays a critical role in regulating drought tolerance in plants, especially its key biosynthetic enzyme, delta-1-pyrroline-5-carboxylate synthase (P5CS), which always positively responds to drought stress. As an important woody oil crop, the expansion of <i>Paeonia ostii</i> cultivation needs to address the issue of plant drought tolerance. Here, we isolated a <i>PoP5CS</i> gene from <i>P. ostii</i>, with an open reading frame of 1842 bp encoding 613 amino acids. <i>PoP5CS</i> expression progressively increased in response to increasing drought stress, and it was localized in the cytoplasm. Silencing of <i>PoP5CS</i> in <i>P. ostii</i> reduced drought tolerance, accompanied by decreased proline content, elevated reactive oxygen species (ROS) accumulation, and increased relative electrical conductivity (REC) and malondialdehyde (MDA) levels. Conversely, overexpression of <i>PoP5CS</i> in <i>Nicotiana tabacum</i> plants enhanced drought resistance, manifested by increased proline levels, reduced ROS accumulation, and lower REC and MDA contents. This study isolates <i>PoP5CS</i> from <i>P. ostii</i> and validates its role in regulating drought tolerance, providing valuable genetic resources and theoretical insights for the development of drought-resistant <i>P. ostii</i> cultivars.