Juvenile hormones (JHs) are farnesoic acid-derived sesquiterpenoids that play a crucial role in regulating various developmental processes in insects. Based on these reported biological activities, JHs and their synthetic analogs have been utilized as insecticides with significant commercial success over the past years. Here we describe the engineering of the JH pathway of the yellow fever mosquito (Aedes aegypti) by transient gene expression in the plant Nicotiana benthamiana. This approach led to the successful production of JH III in N. benthamiana leaves at a concentration of ca. 10 μg/g fresh weight. The co-expression of a feedback-insensitive version of 3-hydroxy-3-methylglutaryl coenzyme A reductase from Arabidopsis thaliana further increased the titer eight-fold from 10 to 80 μg/g fresh weight. Our efforts also revealed that the rich endogenous metabolic background of N. benthamiana can generate farnesoic acid, a key precursor to JH III, and thus, only 3 genes need to be expressed to provide high titers of this compound. Our study demonstrates the production of high titers of JH III in N. benthamina via heterologous expression of insect JH biosynthetic genes.