Saccharides and their derivatives play prominent roles in many primary and secondary metabolic processes. The diversity of their structures and the significance of sugars in diagnosis and treatment of diseases have promoted the fast development of glycobiology. D-sugars, especially D-hexoses have dominated the carbohydrates region, L-hexoses yet are also frequently observed in some important glycoprotein complexes, polysaccharides and antibiotics. Understanding the formation mechanisms of L-hexoses allow rational manipulation of sugar skeletons and further exploration of their applications. L-hexose is usually generated through epimerization of D-hexose by 3,5-epimerase or 5-epimerase. This transformation expands the diversity of sugar configurations and influences the bioactivities of many natural products. The functional and crystallographic studies of 3,5-epimerases and 5-epimerases have provided profound insights into the formation mechanisms of L-hexoses. In this paper, we summarize the catalytic mechanisms of different 3,5-epimerases and 5-epimerases for L-hexose formation. The physiological importance and biomedical prospects of L-hexoses are also discussed.