A full-length cDNA isolated from banana (Musa acuminata L. AAA group) fruit was named MaMDH, containing an open reading frame encoding 332 amino acids that represents the gene for cytoplasmic malic dehydrogenase (MDH). Sequence analysis showed that MaMDH shares high similarity with MDHs from castor bean (XP_002533463), tobacco (CAC12826), peach (AAL11502), and chickpeas (CAC10208). Real-time quantitative polymerase chain reaction (PCR) analysis of MaMDH spatial expression showed that it was expressed in all organs examined: roots, rhizomes, leaves, flowers, and fruits. The expression was the highest in flowers followed by the fruits and roots, whereas the rhizomes and leaves displayed the lowest expression levels. Real-time quantitative PCR revealed that MaMDH exhibited differential expression patterns in post-harvest banana fruits correlating with ethylene biosynthesis. In naturally ripened banana fruits, MaMDH expression was in accordance with ethylene biosynthesis. In accordance, for banana fruits treated with the ethylene analog 1-methylclopropene (1-MCP), MaMDH expression levels were inhibited and remained constant. After treatment with ethylene, MaMDH expression in banana fruits significantly increased with ethylene biosynthesis and peaked 3 days after harvest, which was 11 days earlier than that in naturally ripened banana fruits. These results suggest that MaMDH expression is induced by ethylene to regulate post-harvest banana fruits ripening.