The jasmonate signaling is one of the most studied hormone signaling pathways in plants that mediate important physiological processes. When plants are attacked by herbivores, they induce the biosynthesis of huge pools of jasmonic acid (JA) and the biologically active (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile). JA-Ile is perceived by the receptor complex (SCFCOI1) and facilitates degradation of the repressors of the jasmonate signaling and activation of MYC2 transcription factors (TFs). MYC TFs, then, orchestrate downstream jasmonate responses. Few hours after the initial attack, the levels of JA and JA-Ile wanes to the basal level; however, no mechanism has been identified so far that attenuates defense responses by hydrolysis of JA-Ile. Due of the cost of defense responses, plants regulate the duration and amount of responses. Here, we report two novel regulatory mechanisms that regulate herbivore-induced plant defense responses in N. attenuata. The first regulatory mechanism involves a hydrolase we identified (Jasmonoyl-L-isoleucine hydrolase 1, JIH1) in N. attenuata. JIH1 hydrolyses JA-Ile and attenuates the JA-Ile burst in planta; in a manner that mirrors the in vitro hydrolytic activity of heterologously expressed JIH. When JIH1 was silenced by inverted-repeat silencing, the transformed plants (irJIH1 plants) accumulated significantly more JA-Ile and direct/indirect defenses. The performances of a specialist (Manduca sexta) and generalist (Spodoptera littoralis) herbivores were significantly lower on irJIH1 plants compared to wild type plants. Our data signify the importance of this regulatory mechanism in plant defense regulation.