The annexins are a multifamily of calcium-regulated phospholipid-binding proteins. To investigate the roles of annexins in fiber development, four genes encoding putative annexin proteins were isolated from cotton (Gossypium hirsutum) and designated AnnGh3, AnnGh4, AnnGh5, and AnnGh6. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) results indicated that AnnGh3, AnnGh4, and AnnGh5 were preferentially expressed in fibers, while the transcripts of AnnGh6 were predominantly accumulated in roots. During fiber development, the transcripts of AnnGh3/4/5 genes were mainly accumulated in rapidly elongating fibers. With fiber cells further developed, their expression activity was dramatically declined to a relatively low level. In situ hybridization results indicated that AnnGh3 and AnnGh5 were expressed in initiating fiber cells (0–2 DPA). Additionally, their expression in fibers was also regulated by phytohormones and [Ca²⁺]. Subcellular localization analysis discovered that AnnGh3 protein was localized in the cytoplasm. Overexpression of AnnGh3 in Arabidopsis resulted in a significant increase in trichome density and length on leaves of the transgenic plants, suggesting that AnnGh3 may be involved in fiber cell initiation and elongation of cotton.