The steroid hormone 20-hydroxyecdysone (20E), working through the nuclear receptor heterodimer complex, Ecdysone receptor (EcR) and Ultraspiracle (Usp), triggers key developmental transitions throughout the Drosophila life cycle through genetic regulation. The Sox14 transcription factor has been previously shown to be a primary response gene to the 20E/EcR/Usp complex. We show that animals mutant for sox14 show prepupal and pupal lethality with defects in various 20E developmentally regulated pathways. Northern blot and genome-wide microarray analysis demonstrate that Sox14 is required for the proper expression of both 20E and non 20E-regulated genes at the onset of metamorphosis, expressed in a variety of larval and adult tissues corresponding with the Sox14 expression pattern. Thus, Sox14 is a critical transcription factor required for 20E signaling at the onset of metamorphosis.