Vitamin B₁₂ (B₁₂; also known as cobalamin) is a cofactor in many metabolic processes; deficiency of this vitamin is associated with megaloblastic anaemia and various neurological disorders. In contrast to many prokaryotes, humans and other mammals are unable to synthesize B₁₂. Instead, a sophisticated pathway for specific uptake and transport of this molecule has evolved. Failure in the gastrointestinal part of this pathway is the most common cause of nondietary-induced B₁₂ deficiency disease. However, although less frequent, defects in cellular processing and further downstream steps in the transport pathway are also known culprits of functional B₁₂ deficiency. Biochemical and genetic approaches have identified novel proteins in the B₁₂ transport pathway—now known to involve more than 15 gene products—delineating a coherent pathway for B₁₂ trafficking from food to the body's cells. Some of these gene products are specifically dedicated to B₁₂ transport, whereas others embrace additional roles, which explains the heterogeneity in the clinical picture of the many genetic disorders causing B₁₂ deficiency. This Review describes basic and clinical features of this multistep pathway with emphasis on gastrointestinal transport of B₁₂ and its importance in clinical medicine.