Abstract Strigolactones (SLs) are plant apocarotenoids with diverse functions and structures. The widespread canonical SLs, with distinctive structural variations in their tricyclic lactone known as the ABC-ring, are classified into two types based on the C-ring configurations. The steric C-ring configuration arises during the BC-ring closure downstream of carlactonoic acid (CLA), a biosynthetic intermediate. Most plants stereoselectively produce either type of canonical SLs, e.g., tomato ( Solanum lycopersicum ) produces orobanchol with α-oriented C-ring. The mechanisms governing SL structural diversification are partly understood, with limited insight into the functional implications. Moreover, the precise molecular mechanism for the stereoselective BC-ring closure reaction remains unknown. Herein, we identified an enzyme called the stereoselective BC-ring-forming factor (SRF) from the dirigent protein (DIR) family, especially the DIR-f subfamily, whose biochemical function was previously unidentified, making it a pivotal enzyme in stereoselective canonical SL biosynthesis with the α-oriented C-ring. We begin by confirming the exact catalytic function of the tomato cytochrome P450 SlCYP722C, which we previously demonstrated to be involved in the orobanchol biosynthesis [Wakabayashi et al., Sci. Adv. 5 , eaax9067 (2019)], to convert CLA to 18-oxocarlactonoic acid. Subsequently, we demonstrate that SRF catalyzes the stereoselective BC-ring closure reaction of 18-oxocarlactonoic acid to form orobanchol. Our approach integrates experimental and computational methods, including SRF structure prediction and molecular dynamics simulations, to propose a catalytic mechanism based on the conrotatory 4π-electrocyclic reaction for stereoselective BC-ring formation in orobanchol. The present study provides insight into the molecular basis of how plants produce SLs with specific stereochemistry in a controlled manner.