Background and aims <p>Drought poses a severe threat to the cultivation, yield, and quality of apple, which is an economically important fruit crop. DnaJ proteins, as members of the heat shock protein (HSP) family, play a crucial role in plant responses to abiotic stress. However, the functional characterization of apple DnaJ family genes in conferring drought tolerance remains largely unexplored.</p> Methods <p>In this study, we have performed a genome-wide analysis for <i>DnaJ</i> genes in apple and a candidate gene functional role in drought tolerance was subsequently validated through genetic and physiological assays.</p> Results <p>A total of 111 <i>DnaJ</i> genes were identified in the apple genome and are distributed across 17 chromosomes. Gene structure analysis revealed notable variations in motifs and intron patterns among different <i>DnaJ</i> gene members in apple. Multiple stress-responsive cis-acting elements were identified within the 2kb promoter region upstream of the <i>MdDnaJ</i> genes. RNA-seq analysis revealed that the expression levels of most <i>MdDnaJ</i> genes were induced by drought and salt stress conditions. Subsequent RT-qPCR analysis demonstrated that <i>MdDnaJ107</i> was significantly upregulated under drought stress. Overexpression of <i>MdDnaJ107</i> enhanced drought tolerance in transgenic apple calli, as evidenced by reduced levels of electrolyte leakage, malondialdehyde (MDA), superoxide anion (O₂⁻), and hydrogen peroxide (H₂O₂). In contrast, silencing MdDnaJ107 resulted in the opposite results. In addition, overexpression of <i>MdDnaJ107</i> improved drought tolerance in apple plants, accompanied by enhanced expression of the stress-related genes.</p> Conclusions <p>This study revealed that <i>MdDnaJ107</i> plays a crucial role in conferring drought tolerance, suggesting its potential as a promising target gene for approaches aimed at enhancing plant tolerance to drought stress.</p>