The proteome of 21 DPA (days post-anthesis) of green cotton fiber was analyzed using two-dimensional gel electrophoresis to yield the first reference proteome map. Of 220 individual spots that were excised and analyzed by MALDI-TOF/TOF MS, 156 were identified and cataloged according to their functions. Twelve classes of proteins were identified. Many of these proteins were related to carbohydrate metabolism and energy production. Other notable functional classes included oxidoreductase, cell wall-related and cytoskeleton proteins. This study gives a snapshot of the proteome in 21 DPA green cotton fiber and advances our understanding of molecular mechanisms related to pigment biosynthesis in green cotton fiber.