Cotton (Gossypium hirsutum L.) fibers are single highly elongated cells derived from the outer epidermis of ovules. A large number of genes are required for fiber differentiation and development, but so far, little is known about how these genes control and regulate the process of fiber development. Here we examine the role of the cotton-fiber-specific R2R3 MYB gene GhMYB109 in cotton fiber development. Transgenic reporter gene analysis revealed that a 2-kb GhMYB109 promoter was sufficient to confirm its fiber-specific expression. Antisense-mediated suppression of GhMYB109 led to a substantial reduction in fiber length. Consistently, several genes related to cotton fiber growth were found to be significantly reduced in the transgenic cotton. Our results showed that GhMYB109 is required for cotton fiber development and reveal a largely conserved mechanism of the R2R3 MYB transcription factor in cell fate determination in plants.