Strigolactones (SLs) are important phytohormones that regulate plant architecture, stress response, and adaptation. SLs exuded by roots also act as signals that allow symbiotic fungi and root-parasitic Striga plants to detect their host. Carlactone, the precursor of SLs, is derived from all-trans-β-carotene through the sequential action of the enzymes DWARF27 (D27), carotenoid cleavage dioxygenase 7 (CCD7), and CCD8. D27 catalyses the isomerisation between all-trans and 9-cis-β-carotene, enriching 9-cis-β-carotene, which is the substrate for CCD7. D27 paralogues (D27likes) have also been reported to use 15-cis- or 13-cis-β-carotene isomers as substrates. The molecular basis for the isomerisation of β-carotene by the D27 enzyme family has remained elusive. By using AI-enabled protein structure prediction to guide experimental and computational methods, we demonstrate that D27 contains a 4Fe–4S cluster positioned deep within a hydrophobic cavity that can accommodate β-carotenes. This configuration allows iron cluster–mediated softening of β-carotene through a single-electron transfer reaction and subsequent cavity-induced stereodivergent isomerisation. Differences in cavity dimensions and stereochemistry explain the differences in isomer preference of D27 and D27like1 proteins. Structure-based analysis proposes that the rim areas lining the catalytic site openings of D27, CCD7, and CCD8 immerse into the membrane, implying a mechanism for sequential substrate capture from the membrane, catalysis, and product release into the membrane. Our findings fill a critical gap in the understanding of SL biosynthesis and may inspire new directed interventions to improve plant growth and resilience.