As soil acidification occurs due to industrial and agricultural production processes, it can induce the release of rhizotoxic aluminium ions (Al3+) into the soil, ultimately causing aluminium (Al) stress. Excessive Al content in soil exhibits significant phytotoxicity, inhibiting the growth of roots and stems. In this study, we conducted an investigation into the Al stress tolerance of two apple rootstocks, namely 'YZ3' and 'YZ6', and discovered that 'YZ3' exhibited a superior ability to alleviate the inhibitory effects of Al stress on plant growth. By comparing the transcriptomes of two rootstocks, a differentially expressed gene, MdDUF506, containing an unknown functional (DUF) domain, was identified. Overexpression of MdDUF506 in apple and calli enhances the ability to scavenge reactive oxygen species (ROS), subsequently mitigating the oxidative damage induced by Al stress on plant growth and development. Furthermore, MdDUF506 regulates Al stress tolerance by modulating the expression of genes related to Al stress (MdSTOP1, MdRSL1, MdRSL4, MdGL2, and MdRAE1). MdDUF506 interacts with MdCNR8, positively regulating Al stress tolerance. Taken together, these discoveries offer crucial candidate genes for targeted breeding as well as fresh insights into resistance to Al stress.