MYB transcription factors (TFs) involve in plant abiotic stress tolerance and response in various plant species. In this study, rapid amplification of cDNA ends (RACE) was conducted to isolate the R2R3-MYB TF gene MdSIMYB1 from apples (Malus × domestica). The gene transcripts were abundant in the leaves, flowers and fruits, compared to other organs, and were induced by abiotic stresses and plant hormones. We observed the subcellular localization of an MdSIMYB1-GFP fusion protein in the nucleus. Furthermore, the MdSIMYB1 gene was introduced into the tobacco genome and ectopically expressed in transgenic lines. The results indicate that MdSIMYB1 transgenic tobacco seed germination is insensitive to abscisic acid and NaCl treatment. Additionally, it was found that the ectopic expression of MdSIMYB1 enhanced the tolerance of plants to high salinity, drought and cold tolerance by upregulating the stress-responsive genes NtDREB1A, NtERD10B and NtERD10C. Meanwhile, the transgenic tobacco exhibited robust root growth because of the enhanced expression of the auxin-responsive genes NtIAA4.2, NtIAA4.1 and NtIAA2.5 under stress conditions, which is conducive to stress tolerance. Finally, transgenic apple lines were obtained and tested. Transgenic apple lines that were overexpressing MdSIMYB1 exhibited a higher tolerance to abiotic stress than the wild-type control, but suppression of MdSIMYB1 resulted in lower tolerance. Our results indicate that MdSIMYB1 may be utilized as a target gene for enhancing stress tolerance in important crops.