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Cotton fibers, single seed trichomes derived from ovule epidermal cells, are the major source of global textile fibers. Fiber-specific promoters are desirable to study gene function and to modify fiber properties during fiber development. Here, we revealed that <i>Rho-related GTPase6 (GhROP6)</i> was expressed preferentially in developing fibers. A 1240 bp regulatory region of <i>GhROP6</i>, which contains a short upstream regulatory sequence, the first exon, and the partial first intron, was unexpectedly isolated and introduced into transgenic cotton for analyzing promoter activity. The promoter of <i>GhROP6</i> (<i>proChROP6</i>) conferred a specific expression in ovule surface, but not in the other floral organs and vegetative tissues. Reverse transcription PCR analysis indicated that <i>proGhROP6</i> directed full-length transcription of the fused <i>ß-glucuronidase</i> (<i>GUS</i>) gene. Further investigation of GUS staining showed that <i>proChROP6</i> regulated gene expression in fibers and ovule epidermis from fiber initiation to cell elongation stages. The preferential activity was enriched in fiber cells after anthesis and reached to peak on flowering days. By comparison, <i>proGhROP6</i> was a mild promoter with approximately one-twenty-fifth of the strength of the constitutive promoter <i>CaMV35S</i>. The promoter responded to high-dosage treatments of auxin, gibberellin and salicylic acid and slightly reduced GUS activity under the in vitro treatment. Collectively, our data suggest that the <i>GhROP6</i> promoter has excellent activity in initiating fibers and has potential for bioengineering of cotton fibers.