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The initiation of sex differentiation in insects is regulated by primary sex determination signals. In the Medfly Ceratitis capitataand other Tephritids, Maleness-on-the-Y(MoY) is the master gene for male sex determination. However, the primary signal in Zeugodacus cucurbitae(Coquillett), a very destructive Tephritid pest across the world, remains ambiguous. In this study, we have isolated and characterized the Medfly MoYhomolog in Z. cucurbitae, ZcMoY. ZcMOY protein shows high sequence conservation to its homologs in Bactroceraspecies. ZcMoYtranscription begins and peaks at very early embryonic stages and then becomes undetectable except the testes and heads of day 1 male adults. Silencing ZcMoYin early embryos by RNAi causes abnormal external genitalia and interior reproductive organs, giving rise to intersexes and feminization of XY individuals. The expression pattern and knockdown phenotypes of ZcMoYindicate that ZcMoYplays a key role in regulating sex determination of Z. cucurbitaemales. Our findings will help the understanding of sex determination in Z. cucurbitaeand facilitate the development of genetic sexing strains in its biological control.