Strigolactones are phytohormones that regulate various plant developmental and adaptation processes. When released into soil, strigolactones act as chemical signals, attracting symbiotic arbuscular mycorrhizal fungi and inducing seed germination in root-parasitic weeds. Strigolactones are carotenoid derivatives, characterized by the presence of a butenolide ring that is connected by an enol ether bridge to a less conserved second moiety. Carotenoids are isopenoid pigments that differ in structure, number of conjugated double bonds, and stereoconfiguration. Genetic analysis and enzymatic studies have demonstrated that strigolactones originate from all-trans-β-carotene in a pathway that involves the all-trans-/9-cis-β-carotene isomerase DWARF27 and carotenoid cleavage dioxygenase 7 and 8 (CCD7, 8). The CCD7-mediated, regiospecific and stereospecific double-bond cleavage of 9-cis-β-carotene leads to a 9-cis-configured intermediate that is converted by CCD8 via a combination of reactions into the central metabolite carlactone. By catalyzing repeated oxygenation reactions that can be coupled to ring closure, CYP711 enzymes convert carlactone into tricyclic-ring-containing canonical and non-canonical strigolactones. Modifying enzymes, which are mostly unknown, further increase the diversity of strigolactones. This review explores carotenogenesis, provides an update on strigolactone biosynthesis, with emphasis on the substrate specificity and reactions catalyzed by the different enzymes, and describes the regulation of the biosynthetic pathway.