Although dimorphic sexes have evolved repeatedly in multicellular eukaryotes, their origins are unknown. The mating locus (MT) of the sexually dimorphic multicellular green alga Volvox carteri specifies the production of eggs and sperm and has undergone a remarkable expansion and divergence relative to MT from Chlamydomonas reinhardtii, which is a closely related unicellular species that has equal-sized gametes. Transcriptome analysis revealed a rewired gametic expression program for Volvox MT genes relative to Chlamydomonas and identified multiple gender-specific and sex-regulated transcripts. The retinoblastoma tumor suppressor homolog MAT3 is a Volvox MT gene that displays sexually regulated alternative splicing and evidence of gender-specific selection, both of which are indicative of cooption into the sexual cycle. Thus, sex-determining loci affect the evolution of both sex-related and non-sex-related genes.