Sugar content and cold tolerance are important apple (Malus × domestica) characteristics targeted by breeding programs. Here, a vacuolar membrane sucrose transporter, MdSWEET16, was cloned and characterized. A phylogenetic tree analysis found that MdSWEET16 was on the same polygenetic branch as Arabidopsis thaliana SWEET16 and SWEET17. MdSWEET16 was located on chromosome 2 and consists of six exons and five introns. The recombinant protein was obtained by prokaryotic induction, and the amino acid sequence and transmembrane domain were analyzed by bioinformatics. The encoded ~ 30-kDa protein has seven transmembrane domains and is localized on the tonoplasts of ‘Orin’ callus protoplasts. The expression of MdSWEET16 changed in response to sucrose and low temperature in ‘Orin’ calli. In addition, we also analyzed the expression level of MdSWEET16 during different fruit developmental stages using qRT-PCR. MdSWEET16 was highly expressed in young fruit, and its expression level during fruit development was significantly negatively correlated with sucrose content as assessed by a quantitative fluorescence analysis. Overexpression of MdSWEET16 in ‘Orin’ calli could reduce their sucrose content but increased their cold tolerance compared with MdSWEET16 RNA interference calli, which indicated that MdSWEET16 is involved in the sucrose transport and cold tolerance of apple. MdSWEET16’s expression level was significantly correlated with the sucrose content and low temperature, and was induced by sucrose and low temperatures. Overexpression of MdSWEET16 increased ‘Orin’ calli cold tolerance. The prokaryotic expression of its recombinant protein can be used to further study the function of the MdSWEET16 protein in apple.