Background: Iron (Fe) is indispensable to plants, playing a significant role in life activities such as respiration, chlorophyll biosynthesis, and photosynthetic electron transport. The decrease in pH caused by iron deficiency is related to the activation of H+-ATPase in the root plasma membrane, although the reaction mechanism of this enzyme is not well understood in apples. The H+-ATPase (HA) gene family has been extensively studied in Arabidopsis but is rarely reported in other species. Results: In this study, 14 HA genes were identified from the apple genome database through whole genome analysis. These apple H+-ATPase (MdHAs) genes were classified into four subsets (I, II, IV, V) based on phylogenetic analysis. Bioinformatics analysis revealed that these genes exhibited diversity in gene structure, chromosomal distribution, conserved motifs, and cis-acting elements. The qRT-PCR analysis revealed that iron deficiency stress significantly induced the upregulation of nine MdHA genes (MdHA5-MdHA14). Furthermore, in the roots of iron deficiency-resistant rootstock variety QZ1 compared to the non-resistant variety QZ2, the expression levels of nine genes (including MdHA1, MdHA6-MdHA13) in QZ2 were significantly lower than those in QZ1. This study lays a foundation for further study on the function of HA gene family. Conclusions: These findings suggest that the HA gene family is involved in the apple's response to iron deficiency. This study not only lays the groundwork for future research but also highlights the role of MdHAs in iron stress tolerance. © The Author(s) 2025.