Abstract: 【Objective】 This paper identified the members of transmembrane proton pump pyrophosphatase(H⁺-PPase) gene family in Gossypium hirsutum, and analyzed their expression patterns to provide a theoretical basis for exploring the function, regulation mechanism, and high-quality fiber cotton breeding of this gene family.【Method】 Hidden Markov Model Files(HMM) of the H⁺-PPase gene family(PF03030) were obtained from the Pfam database. G. hirsutum genome was screened for members containing the conserved domains of the H⁺-PPase family. Bioinformatics methods were applied to analyze their physicochemical properties, evolutionary relationships, gene localization, collinearity relationships, gene structure, cisacting elements, and expression patterns. Finally, the expression levels of 10 candidate genes involved in cotton fiber development were detected by using real-time fluorescence quantitative PCR.【Result】 Twenty members of H⁺-PPase gene family(GhH_PPase1-GhH_PPase20) were identified at genome-wide level of G. hirsutum, of which distributed on 12 chromosomes with gene lengths ranging from 2531 to 11810 bp, the number of encoded amino acids residues ranging from 575 to 803, and the protein molecular weight ranging from 60087.76 to 85197.58 Da. All members were hydrophobic and acidic proteins. They could be divided into three major branches:subfamily I(4 members), subfamily IV(4 members) and subfamily V(12 members). Segmental duplication events were the main driving force for the expansion of the G. hirsutum H⁺-PPase gene family. H_PPase1, H_PPase2, H_PPase11 and H_PPase12 on chromosomes A05 and D05 were likely to be the main genes involved in interspecific transmission during evolutionary process. Meanwhile, the collinearity of genes between G. hirsutum and monocot and dicot plants was relatively low, indicating possible independent evolutionary events. There were 14 GhH_PPases proteins contained 10 conserved motifs arranged in the same order, while the remaining six genes showed great differences in their conserved motifs, suggesting potential functional divergence. The promoter regions of the 20 GhH_PPases genes contained numerous light-responsive elements, stress-responsive elements, and hormone-responsive elements. The expression of G. hirsutum H⁺-PPase gene family exhibited temporal and spatial specificity, and more genes insubfamily V played a more important role in cotton fiber development compared to that of other subfamilies.【Conclusion】 The G. hirsutum H⁺-PPase gene family undergoes a certain degree of functional differentiation. Various responsive elements cooperate in growth, development, and responses to adversity. Some genes play important regulatory roles during the elongation and secondary wall thickening stage of G. hirsutum fibers.