The oxylipin plant hormone (3R,7S)‐jasmonoyl‐l‐isoleucine [or (+)‐7‐iso‐jasmonoyl‐l‐isoleucine, JA‐Ile] is widely recognized as a plant defense hormone against pathogens and chewing insects. The metabolism of JA‐Ile into 12‐OH‐JA‐Ile and 12‐COOH‐JA‐Ile is the central mechanism for the inactivation of JA signaling. Recently, 12‐OH‐JA‐Ile was reported to function as a ligand for the JA‐Ile co‐receptor COI1‐JAZ. However, in previous studies, ‘12‐OH‐JA‐Ile’ used was a mixture of four stereoisomers, the naturally occurring cis‐isomer (3R,7S)‐12‐OH‐JA‐Ile and the trans‐isomer (3R,7R)‐12‐OH‐JA‐Ile, and the unnatural cis‐isomer (3S,7R)‐12‐OH‐JA‐Ile and the trans‐isomer (3S,7S)‐12‐OH‐JA‐Ile. Thus, the genuine bioactive form of 12‐OH‐JA‐Ile has not yet been identified. In the present study, we prepared pure stereoisomers of 12‐OH‐JA‐Ile and identified (3R,7S)‐12‐OH‐JA‐Ile as the naturally occurring bioactive form of 12‐OH‐JA‐Ile and found that it binds to COI1‐JAZ9 as effectively as (3R,7S)‐JA‐Ile. In addition, we revealed that the unnatural trans‐isomer (3S,7S)‐12‐OH‐JA‐l‐Ile functions as another bioactive isomer. The pure (3R,7S)‐12‐OH‐JA‐Ile causes partial JA‐responsive gene expression without affecting the expression of JAZ8/10, which is involved in the negative feedback regulation of JA‐signaling. Thus, (3R,7S)‐12‐OH‐JA‐Ile could cause weak and sustainable expression of certain JA‐responsive genes until the catabolism of (3R,7S)‐12‐OH‐JA‐Ile into (3R,7S)‐12‐COOH‐JA‐Ile occurs. The use of chemically pure (3R,7S)‐12‐OH‐JA‐Ile confirmed the genuine biological activities of ‘12‐OH‐JA‐Ile’ by excluding the possible effects of other stereoisomers. A chemical supply of pure (3R,7S)‐12‐OH‐JA‐Ile with an exact bioactivity profile will enable further detailed studies of the unique role of 12‐OH‐JA‐Ile in planta.