Verticillium wilt caused by Verticillium dahliae Kleb is the most destructive disease in cotton‐growing areas around the world, and about 50% of the cotton planting area in China suffers from Verticillium wilt infection, which causes huge economic losses annually. Breeding of broad‐spectrum resistant cotton cultivars through genetic engineering is considered to be one of the most effective approaches to control this fungal disease. We previously reported that a member of Gastrodia antifungal protein (GAFP) family, GAFP4, showed strong antifungal activity to cotton Verticillium wilt. To further enhance the resistance level of transgenic cotton, we supposes that vascular‐specific promoter should be more effective than CaMV 35S promoter. Then we made detailed analysis of the gdcsP promoter from C3‐C4 plant Flaveria anomala and found it contains not only the cis‐acting regulatory elements required for vascular‐ and root‐specific expression, but also disease and wounding inducible elements. The vascular‐specific expression pattern of gdcsP promoter was further confirmed in the transgenic Arabidopsis and cotton expressing β‐glucuronidase (GUS). Moreover, the transgenic cotton plants harboring GAFP4 under control of gdcsP promoter were generated and evaluated for their disease resistance level in Verticillium wilt nursery together with the non‐transgenic control and CaMV 35S:GAFP4 transgenic plants. We demonstrated that with the similar GAFP4 expression, the vascular‐specific expression of GAFP4 plants shows much enhanced resistance to Verticillium wilt. Current data suggested that the vascular‐specific gdcsP promoter is an effective tool not only for genetic engineering of cotton Verticillium wilt resistance, but also for many other applications in genetic engineering.