An essential step in the transmission of the malaria parasite to the Anopheles vector is the transformation of the mature gametocytes into gametes in the mosquito gut, where they egress from the erythrocytes and mate to produce a zygote, which matures into a motile ookinete. Osmiophilic bodies are electron dense secretory organelles of the female gametocytes which discharge their contents during gamete formation, suggestive of a role in gamete egress. Only one protein with no functional annotation, Pfg377, is described to specifically reside in osmiophilic bodies in Plasmodium falciparum. Importantly, Pfg377 defective gametocytes lack osmiophilic bodies and fail to infect mosquitoes, as confirmed here with newly produced pfg377 disrupted parasites. The unique feature of Pfg377 defective gametocytes of lacking osmiophilic bodies was here exploited to perform comparative, label free, global and affinity proteomics analyses of mutant and wild type gametocytes to identify components of these organelles. Subcellular localization studies with fluorescent reporter gene fusions and specific antibodies revealed an osmiophilic body localization for four out of five candidate gene products analyzed: the proteases PfSUB2 (subtilisin 2) and PfDPAP2 (Dipeptidyl aminopeptidase 2), the ortholog of the osmiophilic body component of the rodent malaria gametocytes PbGEST and a previously nonannotated 13 kDa protein. These results establish that osmiophilic bodies and their components are dispensable or marginally contribute (PfDPAP2) to gamete egress. Instead, this work reveals a previously unsuspected role of these organelles in P. falciparum development in the mosquito vector.