Natural rubber (NR), principally comprising cis-1,4-polyisoprene, is an industrially important natural hydrocarbon polymer because of its unique physical properties, which render it suitable for manufacturing items such as tires. Presently, industrial NR production depends solely on latex obtained from the Pará rubber tree, Hevea brasiliensis. In latex, NR is enclosed in rubber particles, which are specialized organelles comprising a hydrophobic NR core surrounded by a lipid monolayer and membrane-bound proteins. The similarity of the basic carbon skeleton structure between NR and dolichols and polyprenols, which are found in most organisms, suggests that the NR biosynthetic pathway is related to the polyisoprenoid biosynthetic pathway and that rubber transferase, which is the key enzyme in NR biosynthesis, belongs to the cis-prenyltransferase family. Here, we review recent progress in the elucidation of molecular mechanisms underlying NR biosynthesis through the identification of the enzymes that are responsible for the formation of the NR backbone structure.