Chitinase is one of the important pathogenesis-related (PR) proteins in plants. By comparative proteomics study, a novel pathogen-responsive chitinase (known as GbCHI) has been identified from sea-island cotton (Gossypium barbadense). The GbCHI cDNA was cloned from wilt-resistant sea-island cotton and the anti-fungal activity of the gene product was investigated. qRT-PCR analysis indicated that GbCHI was expressed constitutively in root, stem, leaf, flower, and ovule of cotton plant, and the expression could be induced by Verticillium dahliae and plant hormone SA, ACC, and JA. Subcellular localization analysis using GFP-tagged proteins showed that GbCHI-GFP fusion proteins were targeted mainly to the plasma membrane. Anti-fungal assay demonstrated that GbCHI could inhibit spore germination and hyphae growth of V. dahliae significantly. These results provide important information for understanding the cellular function of GbCHI and for exploring the application potential of this gene in molecular breeding of wilt-tolerant cotton plants.