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Across species, animals have diverse sex determination pathways, each consisting of a hierarchical cascade of genes and its associated regulatory mechanism. Houseflies have a distinctive polymorphic sex determination system in which a dominant male determiner, the M-factor, can reside on any of the chromosomes. We identified a gene, Musca domestica male determiner (Mdmd), as the M-factor. Mdmd originated from a duplication of the spliceosomal factor gene CWC22 (nucampholin). Targeted Mdmd disruption results in complete sex reversal to fertile females because of a shift from male to female expression of the downstream genes transformer and doublesex. The presence of Mdmd on different chromosomes indicates that Mdmd translocated to different genomic sites. Thus, an instructive signal in sex determination can arise by duplication and neofunctionalization of an essential splicing regulator.