To investigate whether the high expression levels of actin-depolymerizing factor genes are related to pollen development, three GhADF genes (cDNAs) were isolated and characterized in cotton. Among them, GhADF6 and GhADF8 were preferentially expressed in petals, whereas GhADF7 displayed the highest level of expression in anthers, revealing its anther specificity. The GhADF7 transcripts in anthers reached its peak value at flowering, suggesting that its expression is developmentally-regulated in anthers. The GhADF7 gene including the promoter region was isolated from the cotton genome. To demonstrate the specificity of the GhADF7 promoter, the 5'-flanking region, including the promoter and 5'-untranslated region, was fused with the GUS gene. Histochemical assays demonstrated that the GhADF7:GUS gene was specifically expressed in pollen grains. When pollen grains germinated, very strong GUS staining was detected in the elongating pollen tube. Furthermore, overexpression of GhADF7 gene in Arabidopsis thaliana reduced the viable pollen grains and, consequently, transgenic plants were partially male-sterile. Overexpression of GhADF7 in fission yeast (Schizosaccharomyces pombe) altered the balance of actin depolymerization and polymerization, leading to the defective cytokinesis and multinucleate formation in the cells. Given all the above results together, it is proposed that the GhADF7 gene may play an important role in pollen development and germination.