Microtubules undergo dynamic remodeling in response to diverse abiotic stress in plants. The plant-specific IQ67 DOMAIN (IQD) family proteins serve as microtubule-associated proteins, playing multifaceted roles in plant development and response to abiotic stress. However, the biological function of IQD genes in apple remains unclear. In this study, we conducted a comprehensive analysis of the Malus domestica genome, identifying 42 IQD genes distributed across 17 chromosomes and categorized them into four subgroups. Promoter analysis revealed the presence of stress-responsive elements. Subsequent expression analysis highlighted the significant upregulation of MdIQD17 and MdIQD28 in response to cold treatments, prompting their selection for further functional investigation. Subcellular localization studies confirmed the association of MdIQD17 and MdIQD28 with microtubules. Crucially, confocal microscopy and quantification revealed diminished microtubule depolymerization in cells transiently overexpressing MdIQD17 and MdIQD28 compared to wild-type cells during cold conditions. In conclusion, this study provides a comprehensive analysis of IQD genes in apple, elucidating their molecular mechanism in response to cold stress.