Vitamin C (ascorbate) is an excellent and versatile reducing agent, generated from glucose in plants and most animals. In humans and some other species, gulonolactone oxidase, the terminal enzyme in the synthesis pathway, is defective, leaving these species prone to deficiency when dietary intake does not meet metabolic demand. The discovery that ascorbate was required as a cofactor for prolyl hydroxylases integral to collagen synthesis provided a rational explanation for the symptoms of scurvy, the acute vitamin C deficiency disease. These prolyl hydroxylases were the first identified members of the family of iron (Fe)-containing 2-oxoglutarate-dependent dioxygenases (2-OGDDs). Ascorbate supports enzyme activity by maintaining the active site Fe in a reduced state. Many more enzymes belonging to this family have since been identified in plants, animals, and microorganisms. In mammals, more than 50 2-OGDDs have been identified that are involved in many aspects of mammalian biology, including the biosynthesis of carnitine, the posttranslational modification of proteins including histones and the hypoxia-inducible factors, and the modification of DNA and RNAs. Ascorbate is required for the copper (Cu)-containing monooxygenase enzymes that synthesize noradrenalin and amidated neuropeptides, with the potential to impact on mood, vitality, and stress responses. This review discusses the ascorbate requirement of the 2-OGDDs and the Cu-containing monooxygenases and the potential for low ascorbate availability to have an impact on their in vivo activity.