In insects, rapidly evolving primary sex-determining signals are transduced by a conserved regulatory module controlling sexual differentiation. In the agricultural pest (Mediterranean fruit fly, Medfly) we identified a Y-linked gene, (), encoding a small protein that is necessary and sufficient for male development. Silencing or disruption of in XY embryos causes feminization whereas overexpression of in XX embryos induces masculinization. Crosses between transformed XY females and XX males give rise to males and females, indicating that a Y chromosome can be transmitted by XY females. is Y-linked and functionally conserved in other species of the Tephritidae family, highlighting its potential to serve as a tool for developing more effective control strategies against these major agricultural insect pests.