Cotton fiber is a single cell that differentiates from the ovule epidermis and undergoes synchronous elongation with high secretion and growth rate. Apart from economic importance, cotton fiber provides an excellent single-celled model for studying mechanisms of cell-growth. Annexins are Ca(2+)- and phospholipid-binding proteins that have been reported to be localized in multiple cellular compartments and involved in control of vesicle secretions. Although several annexins have been found to be highly expressed in elongating cotton fibers, their functional roles in fiber development remain unknown. Here, 14 annexin family members were identified from the fully sequenced diploid G. raimondii (D5 genome), half of which were expressed in fibers of the cultivated tetraploid species G. hirsutum (cv. YZ1). Among them, GhAnn2 from the D genome of the tetraploid species displayed high expression level in elongating fiber. The expression of GhAnn2 could be induced by some phytohormones that play important roles in fiber elongation, such as IAA and GA3. RNAi-mediated down-regulation of GhAnn2 inhibited fiber elongation and secondary cell wall synthesis, resulting in shorter and thinner mature fibers in the transgenic plants. Measurement with non-invasive scanning ion-selective electrode revealed that the rate of Ca(2+) influx from extracellular to intracellular was decreased at the fiber cell apex of GhAnn2 silencing lines, in comparison to that in the wild type. These results indicate that GhAnn2 may regulate fiber development through modulating Ca(2+) fluxes and signaling.