Technical Abstract: Mannitol, a naturally occurring polyol (sugar alcohol), is widely used in the food, pharmaceutical, medical, and chemical industries. The production of mannitol by fermentation has become attractive because of the problems associated with its production chemically. A number of homo- and heterofermentative lactic acid bacteria, yeasts, and filamentous fungi are known to produce mannitol. In particular, several heterofermentative lactic acid bacteria are excellent producers of mannitol from fructose. These bacteria convert fructose to mannitol with 100% yields from a mixture of glucose and fructose (1:2). Glucose is converted to lactic acid and acetic acid, and fructose is converted to mannitol. The enzyme responsible for conversion of fructose to mannitol is nicotinamide adenine dinucleotide phosphate (NADPH) or nicotinamide adenine dinucleotide (NADH) dependent mannitol dehydrogenase (MDH) and transfers electrons from the reduced forms of these cofactors to fructose. Fructose can also be converted to mannitol by using MDH in the presence of the cofactor nicotinamide adenine dinucleotide phosphate (NADPH) or nicotinamide adenine dinucleotide (NADH). A two enzyme system can be used for cofactor regeneration with simultaneous conversion of two substrates into two products. Mannitol at 180 g/L can be crystallized out from the fermentation broth by cooling crystallization. This paper reviews progress to date in the production of mannitol by fermentation and using enzyme technology, downstream processing, and applications of mannitol.