Abstract: Gastrodia antifungal protein (GAFP) isolated from traditional Chinese medicinal herb Gastrodia elata Bl. is a small protein with a broad spectrum of antifungal activity. As it shows strong resistance to many pathogenic fungi including Vecticillium dahliae kleb and Fusarium oxysporumf. Sp.vasinfectum which cause cotton wilt, it possesses important application potential in plant antifungal genetic engineering. In this study the gene of GAFP (gafp) was transferred into three cultivars of color cotton using the method of pollen-tube-channel. After antifungal selection in the field and molecular assay, we obtained transgenic cotton plants with high level of resistance to wilt. Two Southern-blot-positive plants LB-5-8 and ZB-1-49 showed immunity to wilts. Result of RT-PCR denoted the correct transcription of gafp in LB-5-8 and ZB-1-49. Furthermore, in vitro antifungal activity assay demonstrated that crude protein extraction from them showed obvious resistance to Vecticillium dahliae kleb, which proved the correct expression of gafp in transgenic plants, and suggested the product of translation has antifungal activity. After two years'' breeding, the offspring of these two transgenic plants still showed stable and strong resistance to cotton wilts. Present study provided an effective approach for the prevention and control of cotton wilt by genetic engineering which possess significant applied potential in cotton production.