Sea-island cotton (G. barbadense L.) is an ideal candidate for providing new genetic variations useful to improve fiber quality. The gene expression profile differences of 5, 10, 15, 20, 25DPA fiber between upland cotton and sea-island cotton were analyzed by deep sequencing method (Solexa) with the assembled unigenes as reference sequences. GbPDF1 (PROTODERMAL FACTOR 1) was expressed preferentially during fiber initiation and early elongation. GbPDF1 promoter:GUS constructs in transgenic cotton expressed predominantly in the epidermis of ovules and developing fibers. The 236-bp promoter fragment was sufficient for the basal transcription of GbPDF1 in cotton and the element named HDZIP2ATATHB2 within the fragment was essential for PGbPDF1 expression. PDF1-silenced cotton caused retardation of fiber initiation and produced shorter fibers compared to wild type. Three proteins interacting with GbPDF1 in yeast and in planta might involve in cellular signaling or metabolism. We supposed that GbPDF1 plays a critical role together with the interaction partners in H2O2 homeostasis, steady biosynthesis of ethylene and pectin during fiber development.GbTCP is preferentially expressed in cotton fiber from 5 to 15 DPA. The GbTCP promoter mainly exhibited activities in developing cotton fibre cells and trichomes of Arabidopsis. Constitutive expression of GbTCP in cotton lead to abnormal phenotype including smaller plant, more branches, smaller flowers, leaves and buds, longer flower stalk and lower quality fiber. RNAi suppression of GbTCP gene had no effect on the fiber initiation, but it delayed fiber elongation and induced shorter and lower quality fiber.