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As the main byproduct of cotton fiber, the cotton seed yields about 1.6 times that of fiber, with its oil rich in unsaturated fatty acids, mainly linoleic acid. It is desirable for breeders to increase the oil content of cottonseed without affecting the yield and quality of cotton fiber. In this study, a seed-specific promoter- (alpha-globulin gene promoter-) driven GhDGAT1 overexpression vector (PαGlob-GhDGAT1) was constructed and used to transform an upland cotton line YZ1 (Gossypium hirsutum). Overexpression of the cotton gene GhDGAT1 in cotton seeds increased its total oil content from 4.7% to 13.9% in different transgenic lines and different generations. With the increase of oil content, the composition and contents of the main fatty acids in cotton seed also changed, as reflected by the contents of the main saturated fatty acids and unsaturated oleic acid. GhDGAT1 could be used to increase oil content and improve oil composition in cottonseed.