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Isoprene, emitted by some plants, deters insect herbivory. However, the associated biochemical and physiological responses that confer herbivory resistance remain unknown. We used engineered isoprene-emitting (IE) and non-emitting (NE) control tobacco plants to interpret isoprene-mediated defense against herbivory in plants. Hornworm larvae raised on IE plants exhibited stunted growth compared to those raised on NE plants. Worms preferred to feed on NE rather than IE leaves, indicating deterrent effects of isoprene on insect feeding. Worm feeding induced a greater increase in jasmonic acid (JA), a crucial hormone for insect resistance, in IE leaves compared to that in NE leaves. Assimilation rates were stably maintained in IE plants, suggesting a protective role of isoprene in preserving photosynthetic efficiency during insect herbivory. Wound-induced increase in isoprene emission correlated with the elevation of key metabolites of the isoprene biosynthesis pathway. Our results highlight JA-priming functions of isoprene and provide insights into isoprene-mediated defense against insect herbivory.