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As a great threat to the normal growth of rice, drought not only restricts the growth of rice, but also affects its yield. Glutathione S-transferases (GSTs) have antioxidant and detoxification functions. In rice, GSTs can not only effectively cope with biological stress, but also play a defense role against abiotic stress. In this study, we selected <i>OsGSTU17</i>, a member gene that was induced by drought, to explore the role of GSTs and analyze their physiological mechanisms that are involved in rice drought tolerance. With the CRISPR/Cas9 knockout system techniques, we obtained two independent mutant lines of <i>osgstu17</i>. After 14 days of drought stress treatment, and then re-supply of the water for 10 days, the survival rate of the <i>osgstu17</i> mutant lines was significantly reduced compared to the wild-type (WT). Similarly, with the 10% (<i>w</i>/<i>v</i>) PEG6000 hydroponics experiment at the seedling stage, we also found that compared with the WT, the shoot and root biomass of <i>osgstu17</i> mutant lines decreased significantly. In addition, both the content of the MDA and H₂O₂, which are toxic to plants, increased in the <i>osgtu17</i> mutant lines. On the other hand, chlorophyll and proline decreased by about 20%. The activity of catalase and superoxide dismutase, which react with peroxides, also decreased by about 20%. Under drought conditions, compared with the WT, the expressions of the drought stress-related genes <i>OsNAC10</i>, <i>OsDREB2A</i>, <i>OsAP37</i>, <i>OsP5CS1</i>, <i>OsRAB16C</i>, <i>OsPOX1</i>, <i>OsCATA</i>, and <i>OsCATB</i> in the <i>osgtu17</i> mutant lines were significantly decreased. Finally, we concluded that knocking out <i>OsGSTU17</i> significantly reduced the drought tolerance of rice; <i>OsGSTU17</i> could be used as a candidate gene for rice drought-tolerant cultivation. However, the molecular mechanism of <i>OsGSTU17</i> involved in rice drought resistance needs to be further studied.