Amino acid metabolism plays a crucial role in plant adaptation to drought stress, however, the underlying regulatory mechanisms remain elusive. Here, we identify MdALI, a glutamine amidotransferase in apple, as a new player in drought stress tolerance. Yeast two-hybrid, co-immunoprecipitation, bimolecular fluorescence complementation and split-luciferase assays demonstrate that MdALI interacts with MdMYB88. Biochemical assays confirm its glutamine amidotransferase (GATase) activity, catalyzing the conversion of glutamine to glutamate. Overexpression of MdALI enhances drought tolerance by promoting amino acid accumulation, notably glutamate and proline. Grafting experiments reveal that MdALI-overexpressing rootstocks confer improved drought resilience of grafted plants by modulating amino acid profiles. These findings uncover a GATase-mediated mechanism linking amino acid metabolism to drought tolerance and highlight MdALI as a promising target for breeding drought-tolerance apple rootstocks.