The insect steroid hormone ecdysteroid plays pivotal roles in the temporal coordination of development, represented by molting and metamorphosis. During the larval stages, ecdysteroid is biosynthesized from dietary cholesterol by several ecdysteroidogenic enzymes in the specialized endocrine organ called the prothoracic gland (PG). As ecdysteroid biosynthesis in the PG is affected by several environmental cues, such as photoperiod and nutrition, a fundamental question is how the ecdysteroid biosynthesis pathway is controlled in response to environmental cues. In this review, we briefly summarize recent topics on the regulatory mechanisms of ecdysteroid biosynthesis, especially the neuronal regulatory mechanism, in the fruit fly Drosophila melanogaster. The implications from studies with other insects are also discussed.