Background <p>Pheromones play a key role in regulating sexual behavior throughout the animal kingdom. In <i>Drosophila</i> and other insects, many cuticular hydrocarbons (CHCs) are sexually dimorphic, and some are known to perform pheromonal functions. However, the genetic control of sex-specific CHC production is poorly understood outside of the model species <i>D. melanogaster</i>. A recent evolutionary change is found in <i>D. prolongata</i>, which, compared to its closest relatives, shows greatly increased sexual dimorphism in both CHCs and the chemosensory system responsible for their perception. A key transition involves a male-specific increase in the proportion of long-chain CHCs.</p> Results <p>Perfuming <i>D. prolongata</i> females with the male-biased long-chain CHCs reduces copulation success, suggesting that these compounds function as sex pheromones. The evolutionary change in CHC profiles correlates with a male-specific increase in the expression of multiple genes involved in CHC biosynthesis, including fatty acid elongases, reductases and other key enzymes. In particular, <i>elongase F</i>, which is responsible for producing female-specific pheromones in <i>D. melanogaster</i>, is strongly upregulated in <i>D. prolongata</i> males compared both to females and to males of the sibling species. Mutations in <i>eloF</i> reduce the amount of long-chain CHCs, resulting in a partial feminization of pheromone profiles in <i>D. prolongata</i> males. Transgenic experiments show that sex-biased expression of <i>eloF</i> is caused in part by a putative transposable element <i>honghaier</i> insertion in its regulatory region.</p> Conclusions <p>These results show that <i>cis</i>-regulatory changes in the <i>eloF</i> gene, along with other changes in the CHC synthesis pathway, contribute to the evolution of sexual communication.</p>