SummaryCoumarins are derived via the phenylpropanoid pathway in plants. The 2H‐1‐benzopyran‐2‐one core structure of coumarins is formed via theortho‐hydroxylation of cinnamates,trans/cisisomerization of the side chain, and lactonization.Ortho‐hydroxylation is a key step in coumarin biosynthesis as a branch point from lignin biosynthesis; however,ortho‐hydroxylation of cinnamates is not yet fully understood. In this study, scopoletin biosynthesis was explored usingArabidopsis thaliana, which accumulates scopoletin and its β‐glucopyranoside scopolin in its roots. T‐DNA insertion mutants of caffeoyl CoAO‐methyltransferase 1 (CCoAOMT1) showed significant reduction in scopoletin and scopolin levels in the roots, and recombinant CCoAOMT1 exhibited 3′‐O‐methyltransferase activity on caffeoyl CoA to feruloyl CoA. These results suggest that feruloyl CoA is a key precursor in scopoletin biosynthesis.Ortho‐hydroxylases of cinnamates were explored in the oxygenase families inA. thaliana, and one of the candidate genes in the Fe(II)‐ and 2‐oxoglutarate‐dependent dioxygenase (2OGD) family was designated as F6′H1. T‐DNA insertion mutants ofF6′H1showed severe reductions in scopoletin and scopolin levels in the roots. The pattern ofF6′H1expression is consistent with the patterns of scopoletin and scopolin accumulation. The recombinant F6′H1 protein exhibitedortho‐hydroxylase activity for feruloyl CoA (Km = 36.0 ± 4.27 μm;kcat = 11.0 ± 0.45 sec−1) to form 6′‐hydroxyferuloyl CoA, but did not hydroxylate ferulic acid. These results indicate that Fe(II)‐ and 2‐oxoglutarate‐dependent dioxygenase is the pivotal enzyme in theortho‐hydroxylation of feruloyl CoA in scopoletin biosynthesis.