The Sea Island cotton (Gossypium barbadense L.) has been highly valued in verticillium wilt resistance and many fiber qualities including fiber length, strength and fineness. To identify whether it had some special genes in fiber development in comparison with the Upland cotton (G. hirsutum L.), an actin-depolymerizing factor (ADF) gene was cloned and characterized in this research. A 420 bp open reading frame of the cloned gene, named GbADF1, encoded a protein of 139 amino acids, including 39.57% nonpolar amino acids, 17.27% acidic amino acids, 15.83% basic amino acids and 31.92% hydrophobic amino acids. Its molecular weight was about 15 kDa, and pI 5.04. GbADF1 contained two conserved domains, 6-Ser and the PIP2/actin binding site. Its amino acid sequence was similar to the ADF/cofilin family of other plants. Compared with cDNA sequence, the GbADF1 gene contained one intron near the 3’ end in genomic sequence. Semi-quantitative RT-PCR result showed that GbADF1 was a constitutive expression gene in cotton, and higher expression level was detected in fibers than in trophic tissues. The GbADF1 was successfully expressed as a fusion protein in Escherichia coli BL21 (DE3). The molecular weight was firstly calculated by SDS-PAGE. Western blotting analysis confirmed the existence of a protein corresponding to GbADF1. The structure of GbADF1 was different from that of other ADF genes in higher plant, although the coding sequences of all cloned ADFs were highly conserved.