Juvenile hormones (JHs) are crucial regulators for multiple physiological processes in insects. In the current study, 10 genes in mevalonate pathway involved in JH biosynthesis were identified from Helicoverpa armigera. Tissue-specific expression analysis showed that six genes were highly expressed in the head which contained the JH biosynthetic gland (corpora allata). Temporal expression pattern showed that 10 of 12 genes were highly transcribed in the late 2 nd-instar when the in vivo JH titer reached the peak, indicating a tight correlation between JH titer and the transcription of JH synthetic pathway genes. Moreover, ingestion of methoprene, a JH analogue, significantly suppressed the transcription of nine JH biosynthetic genes and caused a feedback upregulation of the JH degradation enzyme. Particularly, the Acetoacetyl CoA thiolase ( HaAce) and Farnesyl diphosphate synthase gene 4 ( HaFpps4) showed high transcript abundance, and their temporal expressions keep pace with JH fluctuations. Further study by RNAi showed that knockdown of HaFpps4 caused the decrease of JH titer, led to a negative effect on the transcript levels of other genes in JH pathway, and resulted in molting disturbance in larvae. Altogether, these results contribute to our understanding of JH biosynthesis in H. armigera and provide target genes for pest control based on JH-dependent regulation.