Females and males of sexually reproducing animals must cooperate at the molecular and cellular level for fertilization to succeed, even though some aspects of reproductive molecular biology appear to involve antagonistic interactions. We previously reported the existence of a proteolytic cascade in Drosophila melanogaster seminal fluid that is initiated in the male and ends in the female. This proteolytic cascade, which processes at least two seminal fluid proteins (Sfps), is a useful model for understanding the regulation of Sfp activities, including proteolysis cascades in mammals. Here, we investigated the activation mechanism of the downstream protease in the cascade, the astacin-family metalloprotease Seminal metalloprotease-1 (Semp1, CG11864), focusing on the relative contribution of the male and female to its activation. We identified a naturally occurring semp1 null mutation within the Drosophila Genetic Reference Panel. By expressing mutant forms of Semp1 in males homozygous for the null mutation, we discovered that cleavage is required for the complete activation of Semp1, and we defined at least two sites that are essential for this activational cleavage. These amino acid residues suggest a two-step mechanism for Semp1 activation, involving the action of at least two male-derived proteases. Although the cascade's substrates potentially influence both fertility and sperm competition within the mated female, the role of female factors in the activation or activity of Semp1 is unknown. We show here that Semp1 can undergo its activational cleavage in male ejaculates, without female contributions, but that cleavage of Semp1's substrates does not proceed to completion in ejaculates, indicating an essential role for female factors in Semp1's full activity. In addition, we find that expression of Semp1 in virgin females demonstrates that females can activate this protease on their own, resulting in activity that is complete but substantially delayed.