MYB (v-myb avian myeloblastosis viral oncogene homologs) transcription factors (TFs) are involved in diverse physiological processes, including cell shape determination, cell differentiation, and secondary metabolism, as well as abiotic stress response. In the present study, MdMYB4, an R2R3-MYB protein that is a homolog of Arabidopsis thaliana MYB4, was identified and characterized. Quantitative real-time polymerase chain reaction (qRT-PCR) expression analysis demonstrated that MdMYB4 is extensively expressed in various apple (Malus domestica) tissues and that its expression is induced by cold, osmotic, and salt stress. An MdMYB4-GFP fusion protein was localized in the nucleus of transformed onion (Allium cepa) epidermal cells and had a certain transcriptional activation activity by yeast one-hybrid assay. Overexpression of the MdMYB4 gene remarkably enhanced the tolerance of stably transgenic apple calli to severe salt and cold stress, and both the relative conductivity and malondialdehyde (MDA) accumulation of transgenic calli under salt and cold stress were significantly lower than in the wild type control. Taken together, these results suggest that MdMYB4 may play a positive regulatory role in both cold and salt stress responses.