Magnesium (Mg) plays an irreplaceable role in plant growth and development. Mg transporters, especially CorA/MGT/MRS2 family proteins, played a vital role in regulating Mg content in plant cells. Although extensive work has been conducted in model crops, such as Arabidopsis, rice, and maize, the relevant information is scarce in tropical crops. In this study, 10 MaMRS2 genes in banana (Musa acuminata) were isolated from its genome and classified into five distinct clades. The putative physiochemical properties, chromosome location, gene structure, cis-acting elements, and duplication relationships in between these members were analyzed. Complementary experiments revealed that three MaMRS2 gene members (MaMRS2-1, MaMRS2-4, MaMRS2-7), from three distinct phylogenetic branches, were capable of restoring the function of Mg transport in Salmonella typhimurium mutants. Semi-quantitative RT-PCR showed that MaMRS2 genes were differentially expressed in banana cultivar 'Baxijiao' (Musa spp. AAA Cavendish) seedlings. The result was confirmed by real-time PCR analysis, in addition to tissue specific expression, expression differences among MaMRS2 members were also observed under Mg deficiency conditions. These results showed that Mg transporters may play a versatile role in banana growth and development, and our work will shed light on the functional analysis of Mg transporters in banana.