Simple SummaryWe review the role of vitamin B12 in cats. Cobalamin is an essential part of enzymes involved in multiple metabolic reactions. In mammals, two enzymes are cobalamin-dependent: methionine synthase and methylmalonyl-CoA mutase. Cats are obligate carnivores and have an essential requirement for certain nutrients present only in animal tissues, including vitamin B12, which cannot be synthesized by cats. The serum cobalamin concentration can be measured by an automated chemiluminescence competitive binding immunoassay system but does not accurately reflect the real availability of cobalamin, because cobalamin-dependent metabolic reactions occur within cells. A lack of intracellular cobalamin leads to reduced enzyme activity and methylmalonic acid accumulation. The serum cobalamin concentration is particularly useful for detecting deficiency, as it is directly related to the real bioavailability of the vitamin for intracellular enzymatic reactions. Several diseases of the gastrointestinal tract, pancreas, and liver are associated with hypocobalaminemia in cats. Treatment with parenteral cobalamin is always recommended for cats with gastrointestinal disease and low serum cobalamin concentrations. A recognized guideline for cobalamin supplementation does not yet exist for cats, so the recommendations are mainly based on clinical experience.AbstractCobalamin is a water-soluble molecule that has an important role in cellular metabolism, especially in DNA synthesis, methylation, and mitochondrial metabolism. Cobalamin is bound by intrinsic factor (IF) and absorbed in the ileal tract. The IF in cats is synthesized exclusively by pancreatic tissue. About 75% of the total plasma cobalamin in cats is associated with transcobalamin II, while in this species, transcobalamin I is not present. In cats, the half-life of cobalamin is 11–14 days. Diagnostic biomarkers for B12 status in cats include decreased levels of circulating total cobalamin and increased levels of methylmalonic acid. The reference interval for serum cobalamin concentrations in cats is 290–1500 ng/L, and for the serum methylmalonic acid concentration, it is 139–897 nmol/L. Therapy for hypocobalaminemia mainly depends on the underlying disease. In some cases, subcutaneous or intramuscular injection of 250 μg/cat is empirically administered. In recent years, it has been demonstrated that oral cobalamin supplementation can also be used successfully in dogs and cats as a less invasive alternative to parental administration. This review describes the current knowledge regarding B12 requirements and highlights improvements in diagnostic methods as well as the role of hypocobalaminemia in its associated diseases.