A cDNA encoding a cuticle protein containing the R&R Consensus was characterized in the honeybee integument. AmelCPR14 developmental expression is distinguished by an on-off-on pattern, the transition from a low to a high level of transcripts occurring as the ecdysteroid titer is declining after the peak that triggers the onset of pharate (pupal and adult) development. The transcript is abundant during cuticle tanning and sclerotization, and persists even in the adult integument, suggesting that the corresponding protein is required for differentiation and maintenance of the adult cuticle. Such developmental pattern suggested that AmelCPR14 gene might be regulated by the titer of ecdysteroids. We confirmed this hypothesis using different experimental strategies. By tying a ligature in early pupae to prevent exposure of abdominal integument to a high ecdysteroid titer, we delayed the accumulation of AmelCPR14 transcripts in the abdominal integument. This is consistent with ecdysteroid priming being required in pupae for the increase in AmelCPR14 expression in pharate adults. By injecting 20-hydroxyecdysone (20E) in early pupae we demonstrated that hormone titer decay after the peak is critical for AmelCPR14 expression induction. Exposure of pupal integument in vitro to a 20E concentration mimicking the pupal ecdysteroid peak repressed AmelCPR14 expression, which was recovered by hormone removal. Taken together, these data are consistent with an ecdysteroid pulse (increase in hormone titer followed by its decline) being critical for a high AmelCPR14 gene expression in pharate adults.