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Verticillium wilt, mainly caused by a soil-inhabiting fungus <i>Verticillium dahliae</i>, can seriously reduce the yield and quality of cotton. The complex mechanism underlying cotton resistance to Verticillium wilt remains largely unknown. In plants, reactive oxygen species (ROS) mediated by Rbohs is one of the earliest responses of plants to biotic and abiotic stresses. In our previous study, we performed a time-course phospho-proteomic analysis of roots of resistant and susceptible cotton varieties in response to <i>V. dahliae</i>, and found early differentially expressed protein burst oxidase homolog protein D (<i>GhRbohD</i>). However, the role of <i>GhRbohD</i>-mediated ROS in cotton defense against <i>V. dahliae</i> needs further investigation. In this study, we analyzed the function of <i>GhRbohD</i>-mediated resistance of cotton against <i>V. dahliae</i> in vitro and in vivo. Bioinformatics analysis showed that GhRbohD possessed the conservative structural attributes of Rbohs family, 12 members of RbohD out of 57 Rbohs in cotton. The expression of <i>GhRbohD</i> was significantly upregulated after <i>V. dahliae</i> inoculation, peaking at 6 hpi, and the phosphorylation level was also increased. A VIGS test demonstrated that ROS production, NO, H₂O₂ and Ca²⁺ contents of <i>GhRbohD</i>-silenced cotton plants were significantly reduced, and lignin synthesis and callose accumulation were damaged, important reasons for the impairment of <i>GhRbohD</i>-silenced cotton’s defense against <i>V. dahliae</i>. The expression levels of resistance-related genes were downregulated in <i>GhRbohD</i>-silenced cotton by qRT-PCR, mainly involving the lignin metabolism pathway and the jasmonic acid signaling pathway. However, overexpression of <i>GhRbohD</i> enhanced resistance of transgenic <i>Arabidopsis</i> to <i>V. dahliae</i> challenge. Furthermore, Y2H assays were applied to find that GhPBL9 and GhRPL12C may interact with GhRbohD. These results strongly support that <i>GhRbohD</i> activates ROS production to positively regulate the resistance of plants against <i>V. dahliae</i>.