A novel NADP(+)-dependent D-mannitol dehydrogenase and the corresponding gene from the plant symbiotic ascomycete fungus Tuber borchii was identified and characterized. The enzyme, called TbMDH, is a homotetramer with two zinc atoms per subunit. It catalyzed both D-fructose reduction and D-mannitol oxidation, although it showed the highest substrate specificity and catalytic efficiency for D-fructose. Co-factor specificity was restricted to NADP(H) and the reaction proceeded via a sequential ordered Bi Bi mechanism. The carbon responsive transcriptional pattern showed that Tbmdh is up-regulated when mycelia are transferred to a culture medium containing D-mannitol or D-fructose. The phylogenetic analysis showed TbMDH to be the first example of a fungal D-mannitol-2-dehydrogenase belonging to the medium-chain dehydrogenase/reductases (MDRs). The enzyme identified a new group of proteins, most of them annotated in databases as hypothetical zinc-dependent dehydrogenases, forming a distinct subfamily among the polyol dehydrogenase family.