Male-specific exaggerated horns are an evolutionary novelty and have diverged rapidly via intrasexual selection. Here, we investigated the function of the conserved sex-determination gene doublesex (dsx) in the Japanese rhinoceros beetle (Trypoxylus dichotomus) using RNA interference (RNAi). Our results show that the sex-specific T. dichotomus dsx isoforms have an antagonistic function for head horn formation and only the male isoform has a role for thoracic horn formation. These results indicate that the novel sex-specific regulation of dsx during horn morphogenesis might have been the key evolutionary developmental event at the transition from sexually monomorphic to sexually dimorphic horns.