In a full-length cDNA library of roots from Gossypium barbadense Pima90-53 inoculated with Verticillium dahliae, we identified a gene, named GbHyPRP1, which encodes a hybrid proline-rich protein. The nucleotide sequence of the 1747-base pair (bp) cDNA includes a 945 bp open reading frame (ORF), which could encode a protein of 314 amino acids. GbHyPRP1 has a signal peptide, a proline-rich repetitive domain at the N-terminus and a Pollen Ole e I domain at the C-terminus. Homologous analysis found that GbHyPRP1 had a similarity with HyPRP1 proteins from G. raimondii, G. hirsutum and G. arboreum as high as 95.95%, 93.87% and 91.34%, respectively. qRT-PCR (quantitative real time polymerase chain reaction) analysis indicated that the GbHyPRP1 mRNA level was drastically down-regulated in roots of G. barbadense seedlings inoculated with V. dahliae. Furthermore, the ORF of GbHyPRP1 was cloned into the plant expression vector and then introduced into Arabidopsis by Agrobacterium-mediated transformation. Statistical analysis of disease index showed that overexpression of GbHyPRP1 compromised transgenic Arabidopsis plants resistance to V. dahliae. Overall, our results suggest that GbHyPRP1 involves in cotton resistance to V. dahliae and may be an important negative regulator.