Hydroxycinnamoyl CoA: Shikimate/quinate hydroxycinnamoyl transferase (HCT) is a key enzyme involved in the lignin biosynthesis pathway, which affects the biosynthesis of G/S monolignol. In cotton fibers, the content of lignin is closely related to fiber quality. A full- length gene from brown cotton (Gossypium hirsutum L.) cultivar Xincaimian 6 fiber at the 16 day post anthesis (16 DPA) was cloned encoding HCT ortholog, which was named GhHCT (GenBank Accession No. KF749428). The expression of GhHCT in different tissue and organs were examined through Real-time PCR technology. The full-length of the GhHCT ortholog consisted of a 1 500 bp open reading frame (ORF) encoding a polypeptide containing 499 amino acid residues. The molecular weight of deduced amino acids was 55.2 kD, with an isoelectric point (pl) of 5.69. The deduced amino acid sequence showed 82%?95% identities with HCTs of other plants. The deduced amino acid sequence of the GhHCT ortholog has a histidine containing motif (HHAAD), characteristic for acyl transfer catalysis. A second consensus sequence, a DFGWG block, was another acyl transferase of the BAHD gene family. Phylogenetic analysis showed the closest relationship (94.9%) with HCT of Hibiscus cannabinus (HcHCT). According to quantitative Real- time reverse transcription PCR (qRT- PCR) analysis, GhHCT transcript expressed in all the tissues and organs, but the highest expression was observed in ovules at 16 DPA. A relatively high expression was also detected in flowers and stems. The lowest level of expression of GhHCT was found in leaves. This study provides the crucial information for further study on the biological functions of GhHCT gene and molecular mechanism of lignin biosynthesis in cotton fiber. The study also provides clues and theoretical basis for improving fiber quality of naturally colored cotton through genetic engineering.