Cytokinins (CK) are a group of phytohormones that are well-known to regulate the plant development, but also environmental interactions. The most prominent examples for CK functions in plant-insect interactions are the leaf miner and sawfly induced formation of “green islands” and leaf galls, respectively. Aside from the abuse by insects, CKs were also indicated to up-regulate plant defenses, although much information is still missing. In this work I used the wild tobacco Nicotiana attenuata and its natural occurring herbivore Manduca sexta to analyze the interaction between the CK pathway and the herbivory-induced defense responses. Wounding and the perception of specific herbivore-derived elicitors induced changes in various parts of the CK-pathway of N. attenuata. This included the transcript-levels of genes involved in CK biosynthesis, degradation and signaling, as well as the abundance of CK metabolites, such as isopentenyladenosine. Interestingly, jasmonic acid (JA), a key regulator of the herbivory-induced plant response, was not necessary for these changes, but even attenuated some responses. To analyze the effects of the CK pathway on N. attenuata’s defense chemistry I used multiple technics to manipulate its CK levels and signaling, including external CK application, chemically-inducible expression of a CK biosynthesis gene (isopentenyltransferase), as well as transient and constitutive silencing of the CK receptors, NaCHK2 and NaCHK3. The CK pathway was found as positive regulator of the herbivory-induced accumulation of JA, but not of its bioactive JA-isoleucine conjugate. Still, a functional CK pathway was found as prerequisite for a strong induction of anti-herbivore defenses, such as the phenolamide caffeoylputrescine or proteinase inhibitors. Additionally, CK levels influenced the interaction of N. attenuata with the mirid bug, Tupiocoris notatus and also the CK pathway of the model plant Arabidopsis thaliana responded to herbivory.