Plant hybrid proline-rich proteins (HyPRPs) usually consist of an N-terminal signal peptide, a central proline-rich domain, and a conserved eight-cysteine motif C-terminal domain. In this study, one gene (designated as GhHyPRP4) encoding putative HyPRP was isolated from cotton cDNA library. Northern blot and quantitative reverse transcriptase-polymerase chain reaction analyses revealed that GhHyPRP4 was preferentially expressed in leaves. Under cold stress, GhHyPRP4 expression was significantly up-regulated in leaves of cotton seedlings. Using the genome walking approach, a promoter fragment of GhHyPRP4 gene was isolated from cotton genome. GUS (β-glucuronidase) gene driven by GhHyPRP4 promoter was specifically expressed in leaves and cotyledons of the transgenic Arabidopsis thaliana. Furthermore, GUS expression in leaves was remarkably induced by cold stress. Overexpression of GhHyPRP4 in yeast (Schizosaccharomyces pombe) significantly enhanced the cell survival rate upon treatment under -20°C for 60 h. These data suggested that GhHyPRP4 may be involved in plant response to cold stress during seedling development of cotton.