Homeodomain-leucine zipper (HD-Zip) proteins are transcriptional factors involved in plant development. In this study, one cDNA clone (Gossypium hirsutum homeobox1, designated GhHB1) encoding HD-Zip protein was isolated from a cotton root cDNA library. The GhHB1 cDNA is 1132 bp in length, including an 828 bp open reading frame that encodes a peptide with 275 amino acids, and 5′-/3′-untranslated regions. The predicted GhHB1 protein containing a homeodomain and a leucine-rich zipper motif shares relatively high identity with other plant HD-Zip proteins. Analysis using quantitative real-time RT-PCR indicated that the GhHB1 gene is predominantly expressed in roots and hypocotyls. Furthermore, GhHB1 transcripts were largely accumulated in early root development, and significantly reduced to very low levels as roots further developed, suggesting that the gene might function in the early development of roots. Under treatment with 1% NaCl, the expression level of the GhHB1 gene was dramatically increased in roots. Likewise, GhHB1 activity in roots was up-regulated by abscisic acid. These results imply that GhHB1 might play an important role in response to salt stress and to abscisic acid signaling.