Verticillium wilt, caused by , is a devastating disease for many important crops, including cotton. Kiwellins (KWLs), a group of cysteine-rich proteins synthesized in many plants, have been shown to be involved in response to various phytopathogens. To evaluate genes for their function in resistance to Verticillium wilt, we investigated KWL homologs in cotton. Thirty-five genes () were identified from the genome of upland cotton (). Among them, GhKWL1 was shown to be localized in nucleus and cytosol, and its gene expression is induced by the infection of . We revealed that GhKWL1 was a positive regulator of . Silencing of resulted in a decrease, whereas overexpression led to an increase in resistance of transgenic plants to Verticillium wilt. Interestingly, through binding to GhKWL1, the pathogenic effector protein VdISC1 produced by could impair the defense response mediated by GhKWL1. Therefore, our study suggests there is a GhKWL1-mediated defense response in cotton, which can be hijacked by through the interaction of VdISC1 with GhKWL1.