Carotenoid pigments produce most red, orange and yellow colours in vertebrates. This coloration can serve as an honest signal of quality that mediates social and mating interactions, but our understanding of the underlying mechanisms that control carotenoid signal production, including how different physiological pathways interact to shape and maintain these signals, remains incomplete. We investigated the role of testosterone in mediating gene expression associated with a red plumage sexual signal in red-backed fairywrens (Malurus melanocephalus). In this species, males within a single population can flexibly produce either red/black nuptial plumage or female-like brown plumage. Combining correlational analyses with a field-based testosterone implant experiment and qPCR, we show that testosterone mediates expression of carotenoid-based plumage in part by regulating expression of CYP2J19, a ketolase gene associated with ketocarotenoid metabolism and pigmentation in birds. This is the first time that hormonal regulation of a specific genetic locus has been linked to carotenoid production in a natural context, revealing how endocrine mechanisms produce sexual signals that shape reproductive success.