BACKGROUND Molybdenum (Mo) is an essential micronutrient for almost all biological systems, which holds key positions in several enzymes involved in carbon, nitrogen and sulfur metabolism. In general, this transition metal needs to be coordinated to a unique pterin, thus forming a prosthetic group named molybdenum cofactor (Moco) at the catalytic sites of molybdoenzymes. The biochemical functions of many molybdoenzymes have been characterized; however, comprehensive analyses of the evolution of Mo metabolism and molybdoproteomes are quite limited. RESULTS In this study, we analyzed almost 5900 sequenced organisms to examine the occurrence of the Mo utilization trait at the levels of Mo transport system, Moco biosynthetic pathway and molybdoproteins in all three domains of life. A global map of Moco biosynthesis and molybdoproteins has been generated, which shows the most detailed understanding of Mo utilization in prokaryotes and eukaryotes so far. Our results revealed that most prokaryotes and all higher eukaryotes utilize Mo whereas many unicellular eukaryotes such as parasites and most yeasts lost the ability to use this metal. By characterizing the molybdoproteomes of all organisms, we found many new molybdoprotein-rich species, especially in bacteria. A variety of new domain fusions were detected for different molybdoprotein families, suggesting the presence of novel proteins that are functionally linked to molybdoproteins or Moco biosynthesis. Moreover, horizontal gene transfer event involving both the Moco biosynthetic pathway and molybdoproteins was identified. Finally, analysis of the relationship between environmental factors and Mo utilization showed new evolutionary trends of the Mo utilization trait. CONCLUSIONS Our data provide new insights into the evolutionary history of Mo utilization in nature.