The biosynthesis of the molybdenum cofactor (Moco) is ubiquitous and highly conserved in all organisms from bacteria to humans. In Moco, the molybdenum atom is coordinated to a dithiolene group present in the pterin-based 6-alkyl side chain of molybdopterin (MPT). In general, the biosynthesis of Moco can be divided into three steps in eukaryotes, and four steps in bacteria and archaea: (i) the starting point is the formation of the cPMP from 5′GTP, (ii) in the second step MPT is formed by the insertion of two sulfur molecules into cPMP, (iii) in the third step the molybdenum atom is inserted into MPT and Moco is formed, and (iv) additional modification of Moco occurs in bacteria and archaea in a fourth step by the attachment of nucleotides (CMP or GMP) to the phosphate group of MPT, forming the dinucleotide variants of Moco. Further, small differences exist in Moco formation among the different phyla. In higher eukaryotes Moco biosynthesis is located in different cellular compartments, many individual Moco biosynthesis proteins appear to have several cellular roles, and some proteins are shared between different biosynthetic pathways. Further, bacteria contain a large variety of more than 60 different molybdoenzymes being involved in specific, however, usually nonessential redox reactions. In contrast, in humans only four different molybdoenzymes have been identified, and a defect in Moco biosynthesis is lethal due to the loss of sulfite oxidase activity. This review will focus on the biosynthesis of Moco in bacteria and humans.