Sulfur is an essential nutrient for plant growth and development. Sulfate transporters (Sultrs) are critical for sulfate () uptake from the soil by the roots in higher plants. However, knowledge about Sultrs in apples (Malus domestica) is scarce. Here, nine putative MdSultrs were identified and classified into two groups according to the their phylogenetic relationships, gene structures, and conserved motifs. Various cis-regulatory elements related to abiotic stress and plant hormone responsiveness were found in the promoter regions of MdSultrs. These MdSultrs exhibited tissue-specific expression patterns and responded to low sulfur (S), abscisic acid (ABA), indole-3-acetic acid (IAA), and methyl jasmonate (MeJA), wherein MdSultr3;1a was especially expressed in the roots and induced by low S. The uptake of in cultivated apples depends on the roots of its rootstock, and MhSultr3;1a was isolated from Malus hupehensis roots used as a rootstock. MhSultr3;1a shared 99.85% homology with MdSultr3;1a and localized on the plasma membrane and nucleus membrane. Further function characterization revealed that MhSultr3;1a complemented an transport-deficient yeast mutant and improved the growth of yeast and apple calli under low S conditions. The MhSultr3;1a-overexpressing apple calli had a higher fresh weight compared with the wild type (WT) under a low-S treatment because of the increased and cysteine (Cys) content. These results demonstrate that MhSultr3;1a may increase the content of and Cys to meet the demands of S-containing compounds and improve their growth under S-limiting conditions.