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Cotton is an important economic crop. Cotton Verticillium wilt caused by <i>Verticillium dahliae</i> seriously damages production. Phytosterols play roles in plant-pathogen interaction. To explore the function and related mechanism of phytosterols in the interaction between <i>Verticillium dahliae</i> and cotton plants, and the resistance to Verticillium wilt, in this study, we analyzed the changes of sterol composition and content in cotton roots infected by <i>Verticillium dahliae</i>, and identified the sterol C22-desaturase gene <i>GhCYP710A1</i> from upland cotton. Through overexpressing and silencing the gene in cotton plant, and ectopically expressing the gene in <i>Arabidopsis</i>, we characterized the changes of sterol composition and the resistance to Verticillium wilt in transgenic plants. The infection of <i>Verticillium dahliae</i> resulted in the content of total sterol and each sterol category decreasing in cotton root. The ratio of stigmasterol to sitosterol (St/Si) increased, indicating that the conversion of sitosterol to stigmasterol was activated. Consistently, the expression level of <i>GhCYP710A1</i> was upregulated after infection. The GhCYP710A1 has the conservative domain that is essential for sterol C22-desaturase in plant and is highly expressed in root and stem, and its subcellular location is in the endoplasmic reticulum. The ectopic expression of <i>GhCYP710A1</i> gene promoted the synthesis of stigmasterol in <i>Arabidopsis</i>. The St/Si value is dose-dependent with the expression level of <i>GhCYP710A1</i> gene. Meanwhile, the resistance to Verticillium wilt of transgenic <i>Arabidopsis</i> increased and the permeability of cell membrane decreased, and the content of ROS decreased after V991 (a strain of <i>Verticillium dahliae</i>) infection. Consistently, the resistance to Verticillium wilt significantly increased in the transgenic cotton plants overexpressing <i>GhCYP710A1</i>. The membrane permeability and the colonization of V991 strain in transgenic roots were decreased. On the contrary, silencing <i>GhCYP710A1</i> resulted in the resistance to Verticillium wilt being decreased. The membrane permeability and the colonization of V991 were increased in cotton roots. The expression change of <i>GhCYP710A1</i> and the content alteration of stigmasterol lead to changes in JA signal transduction, hypersensitivity and ROS metabolism in cotton, which might be a cause for regulating the Verticillium wilt resistance of cotton plant. These results indicated that <i>GhCYP710A1</i> might be a target gene in cotton resistance breeding.