Cotton fiber elongation, largely achieved by cell wall loosening, is an important stage during cotton fiber development. In this present research, a fiber preferential cDNA encoding a pectate lyase (PEL) which could exclusively degrade the de-esterified pectin was isolated from a cotton (Gossypium hirsutum) fiber cDNA library. Subsequently, the corresponding PEL genes were isolated from four different cotton species and characterized. In vitro enzyme assays indicated that GhPEL really exhibited cleavage-activity against de-esterified pectin. The temporal-spatial expression analyses revealed that the GhPEL gene was preferentially expressed in fibers at 10 days-post anthesis (DPA). Antisense GhPEL transgenic cotton plants were generated by Agrobacterium-mediated transformation. Six homozygous lines, each with one or two copies of the transgene inserted as determined by southern blot analysis of the NPTII gene, were selected for further functional analysis. The GhPEL expression during fiber elongation in these transgenic lines was significantly suppressed in various degrees. Furthermore, the reduction of GhPEL enzymatic activity by decreasing GhPEL transcripts severely affected the degradation of de-esterified pectin in primary cell walls of transgenic cotton fibers, which consequently blocked cell wall loosening in early fiber development. Ultimately, the fiber elongation of all these transgenic lines was repressed. These results suggested that GhPEL may play an important role in the process of normal fiber elongation in cotton.