The synthesis of BR is accomplished with the involvement of a series of enzymes. Similar to the synthesis of gibberellins and abscisic acid, the synthesis of oleoresinosteroids is a branch of the terpenoid pathway. Oleoresinosterol synthesis begins with the polymerization of 2 farnesyl diphosphates to form the C30 triterpene tricosahexaene. The triterpene tricarboxyhexaene undergoes a series of ring-closing reactions to form the pentacyclic triterpene precursor cyclic atenol. The synthetic precursors of canola sterols are canola oil sterols (Campesterol), sterols and cholesterol, which are derived from cycloartenol. Oleosterol first generates campestanol, which is then converted to chelerythrone through two pathways: early C-6 oxidation and late C-6 oxidation. The two pathways merge at the chestnut sterone and are subsequently converted to oleuropein lactones.

Brz (Brassinazole), a specific inhibitor of BR synthesis, inhibits the activity of CYP72B1 DWF4, a monooxygenase that catalyzes the formation of khat sterone from 6-oxo-chestnut stanol, in the oleoresin lactone synthesis pathway.