Updated project metadata. Fertility depends, in part, on interactions between male and female reproductive proteins inside the female reproductive tract (FRT) that mediate postmating changes in female behavior, morphology, and physiology. Coevolution between interacting proteins within species may drive reproductive incompatibilities between species, yet the mechanisms underlying postmating-prezygotic isolating barriers remain poorly resolved. Here, we used quantitative proteomics in sibling Drosophila species to investigate the molecular composition of the FRT environment and its role in mediating species-specific postmating responses. We found that (1) FRT proteomes in D. simulans and D. mauritiana virgin females express unique combinations of secreted proteins and are enriched for distinct functional categories, (2) mating induces substantial changes to the FRT proteome in D. mauritiana but not in D. simulans, and (3) the D. simulans FRT proteome exhibits limited postmating changes irrespective of whether females mate with conspecific or heterospecific males, suggesting an active female role in mediating reproductive interactions. Our study suggests that divergence in the FRT extracellular environment and postmating response contribute to previously described patterns of postmating-prezygotic isolation and the maintenance of species boundaries.