Gametocytes, the precursor cells of malaria-parasite gametes, circulate in the blood and are responsible for transmission from host to mosquito vector. The individual proteomes of male and female gametocytes were analyzed using mass spectrometry, following separation by flow sorting of transgenic parasites expressing green fluorescent protein, in a sex-specific manner. Promoter tagging in transgenic parasites confirmed the designation of stage and sex specificity of the proteins. The male proteome contained 36% (236 of 650) male-specific and the female proteome 19% (101 of 541) female-specific proteins, but they share only 69 proteins, emphasizing the diverged features of the sexes. Of all the malaria life-cycle stages analyzed, the male gametocyte has the most distinct proteome, containing many proteins involved in flagellar-based motility and rapid genome replication. By identification of gender-specific protein kinases and phosphatases and using targeted gene disruption of two kinases, new sex-specific regulatory pathways were defined.