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Jasmonic acid (JA) plays important roles in plant resistance to insect herbivores. One important derivative of JA is 12-OH-JA, which is produced by two independent pathways: direct hydroxylation of JA by jasmonate-induced oxygenases (JOXs) or hydrolyzation of 12-OH-JA-Ile.Yet the function of 12-OH-JA in plant-herbivore interactions remains largely unknown. In this study, we silenced four JOX homologs independently in the wild tobacco Nicotiana attenuata by virus-induced gene silencing (VIGS), and found that all four JOX homologs are involved in JA hydroxylation. Simultaneously silencing the four JA hy-droxylases in VIGS-NaJOXs plants decreased herbivory-induced 12-OH-JA by 33％, but JA and JA-Ile levels increased by 45％ and 30％, respectively, compared to those in control plants. Compared to direct hy-droxylation from JA, hydrolyzation from 12-OH-JA-Ile is equally important for herbivory-induced 12-OH-JA accumulation:in the 12-OH-JA-Ile deficient irJAR4/6 plants, 12-OH-JA decreased 34％. Moreover, VIGS-NaJOXs plants exhibited enhanced resistance to the generalist herbivore Spodoptera litura. The poor larval performance was strongly correlated with high levels of several JA-Ile-dependent direct defense me-tabolites in the VIGS-NaJOXs plants. When we simultaneously silenced all four JA hydroxylases in the JA-Ile-deficient irJAR4/6 background, the enhanced herbivore resistance diminished, demonstrating that enhanced herbivore resistance resulted from elevated JA-Ile levels. Given that silencing these NaJOX-like genes did not detectably alter plant growth but highly increased plant defense levels, we propose that JOX genes are potential targets for genetic improvement of herbivore-resistant crops.