D-mannitol is a kind of hexitols widely applied in the food and medicinal fields due to its numerous benefits. Mannitol 2-dehydrogenase (MDH, EC 1.1.1.67) is a kind of oxidoreductase playing a pivotal part in the production of D-mannitol from D-fructose. In this work, we identified a highly thermostable D-mannitol-producing MDH from a thermo-tolerant bacterium, Caldicellulosiruptor morganii Rt8.B8. When using D-fructose as the substrate, the recombinant MDH was activated obviously in the presence of Mn2+ with an optimal pH as 8.0 and temperature at 75 degrees C. The specific activity, Michaelis-Menten constant (K-m) and catalytic efficiency (k(cat)/K-m) for D-fructose were determined as 115 U mg(-1), 18 mM and 8.5 s(-1) mM(-1). Moreover, the half-life (t(1/2)) of recombinant MDH at 75, 85 and 95 degrees C was 19 h, 3.5 h and 1.62 h respectively, which was much higher than that of most MDHs. The optimal condition for the production of D-mannitol was determined to be pH at 7.5, the temperature at 70 degrees C, and 2:1 ratio of C. morganii MDH and Ogataea parapolymotpha formate dehydrogenase (FDH, EC 1.2.1.2). Meanwhile, approximately 80 % D-mannitol was generated by two enzymes after a 50 h reaction from 400 mM D-fructose, indicating a great potentiality in the industrial preparation of D-mannitol.