Obesity, diabetes, and other cardiovascular diseases are directly related to the high consumption of processed sugars with high caloric content. The current food industry has novel trends related to replacing highly caloric sugars with non-caloric or low-calorie sweeteners. Mannitol, a polyol, represents a suitable substitute because it has a low caloric content and does not induce a glycemic response, which is crucial for diabetic people. Consequently, this polyol has multiple applications in the food, pharmaceutical, and medicine industries. Mannitol can be produced by plant extraction, chemical or enzymatic synthesis, or microbial fermentation. Different in vitro processes have been developed regarding enzymatic synthesis to obtain mannitol from fructose, glucose, or starch-derived substrates. Various microorganisms such as yeast, fungi, and bacteria are applied for microbial fermentation. Among them, heterofermentative lactic acid bacteria (LAB) represent a reliable and feasible alternative due to their metabolic characteristics. In this regard, the yield and productivity of mannitol depend on the culture system, the growing conditions, and the culture medium composition. In situ mannitol production represents a novel approach to decrease the sugar content in food and beverages. Also, genetic engineering offers an interesting option to obtain mannitol-producing strains. This review presents and discusses the most significant advances that have been made in the mannitol production through fermentation by heterofermentative LAB, including the pertinent and critical analysis of culture conditions considering broth composition, reaction systems, and their effects on productivities and yields.