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MAIN CONCLUSION The discovery of three iridoid synthases (GjISY, GjISY2 and GjISY4) from Gardenia jasminoides and their functional characterization increase the understanding of iridoid scaffold/iridoid glycoside biosynthesis in iridoid-producing plants. Iridoids are a class of noncanonical monoterpenes that are found naturally in the plant kingdom mostly as glycosides. Over 40 iridoid glycosides (e.g., geniposide, gardenoside and shanzhiside) have been isolated from Gardenia jasminoides. They have multiple pharmacological properties and health-promoting effects. However, their biosynthetic pathway is poorly understood, and the iridoid synthase (ISY) responsible for the cyclization of the core scaffold remains unclear. In this study, three homologs of ISYs from G. jasminoides (GjISY, GjISY2 and GjISY4) were identified on the basis of transcriptomic data and functionally characterized. The genomic structure and intron-exon arrangement revealed that all three ISYs contained an intron. Biochemical assays indicated that all three recombinant enzymes reduced 8-oxogeranial to nepetalactol and its open forms (iridodials) as the products of the classical CrISY (Catharanthus roseus). In addition, all three enzymes reduced progesterone to 5-β-prognane-3,20-dione. However, only GjISY2 and GjISY4 reduced 2-cyclohexen-1-one to cyclohexanone. Overall, the GjISY2 expression levels in the flowers and fruits were similar to the GjISY and GjISY4 expression levels. By contrast, the GjISY2 expression levels in the upper and lower leaves were substantially higher than the GjISY and GjISY4 expression levels. Among the three, GjISY2 exhibited the highest catalytic efficiency for 8-oxogeranial. GjISY2 might be the major contributor to iridoid biosynthesis in G. jasminoides. Collectively, our results advance the understanding of iridoid scaffold/iridoid glycoside biosynthesis in G. jasminoides and provide a potential target for metabolic engineering and breeding.