The genomes of malaria parasites (Plasmodium spp.) contain a family of genes encoding proteins with a Plasmodium helical interspersed subtelomeric (PHIST) domain, most of which are predicted to be exported into the parasite-infected human red blood cell (iRBC). Here, using transgenic parasites and a combination of cellular, biochemical, and biophysical assays, we have characterized and determined the function of a novel member of the PHIST protein family in Plasmodium falciparum, termed lysine-rich membrane-associated PHISTb (LyMP). LyMP was shown to associate directly with the cytoskeleton of iRBCs where it plays a role in their abnormal ability to adhere to a protein expressed on vascular endothelial cells, resulting in sequestration. Deletion of LyMP dramatically reduced adhesion of iRBCs to CD36 by 55%, which was completely restored to wild-type levels on complementation. Intriguingly, in the absence of LyMP, formation of RBC membrane knobs and the level of surface exposure of the parasites' major cytoadhesive ligand, PfEMP1, were identical to those for the parental parasite line, demonstrating for the first time an additional mechanism that enhances cytoadherence of iRBCs beyond those already recognized. Our findings identify LyMP as a previously unknown RBC cytoskeletal-binding protein that is likely to be of major significance in the complex pathophysiology of falciparum malaria.-Proellocks, N. I., Herrmann, S., Buckingham, D. W., Hanssen, E., Hodges, E. K., Elsworth, B., Morahan, B. J., Coppel, R. L., Cooke, B. M. A lysine-rich membrane-associated PHISTb protein involved in alteration of the cytoadhesive properties of Plasmodium falciparum infected red blood cells.