The malaria parasite Plasmodium falciparum exports several hundred proteins into 
the infected erythrocyte that are involved in cellular remodeling and severe 
virulence. The export mechanism involves the Plasmodium export element (PEXEL), 
which is a cleavage site for the parasite protease, Plasmepsin V (PMV). The PMV 
gene is refractory to deletion, suggesting it is essential, but definitive proof 
is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks 
the activity of PMV isolated from P. falciparum and Plasmodium vivax. Assessment 
of PMV activity in P. falciparum revealed PEXEL cleavage occurs 
cotranslationaly, similar to signal peptidase. Treatment of P. 
falciparum-infected erythrocytes with the inhibitor caused dose-dependent 
inhibition of PEXEL processing as well as protein export, including impaired 
display of the major virulence adhesin, PfEMP1, on the erythrocyte surface, and 
cytoadherence. The inhibitor killed parasites at the trophozoite stage and 
knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of 
PMV increased resistance. This provides the first direct evidence that PMV 
activity is essential for protein export in Plasmodium spp. and for parasite 
survival in human erythrocytes and validates PMV as an antimalarial drug target.