Mannitol-2-dehydrogenase (MtDH) (E.C. 1.1.1.67) gene was cloned from Thermotoga neapolitana DSM 4359 and expressed in Escherichia coli BL21. The purified enzyme showed a predicted clear band of 36kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), native molecular mas was 135kDa. Km and Vmax values for reduction of D-fructose to D-mannitol were 20mM and 200U mg-1 respectively. kcat for reduction direction was 180s−1 and kcat/Km were 9mM−1s−1. The enzyme showed optimal pH at 6.5 and the optimum temperature was 90°C with 100% relative activity. The purified enzyme was quite stable at 75°C and had half of initial activity after 1h of incubation at 90°C. (TnMtDH) showed no activity with xylitol, inositol, sorbitol, rahmanose, mannose and xylose, and with NADPH and NADP+ as co factors. The presence of some divalent metals in the reaction enhanced the enzyme activity. The enzyme might be utilizing to produce mannitol without other sugar conformation under high temperature.