Malaria is caused by protozoan parasites, Plasmodium spp., that belong to the phylum Apicomplexa. The life cycle of these parasites depends on two different hosts; the definitive host, or vector, is a mosquito, and the intermediate host is a vertebrate, such as human. Malaria parasites use a unique form of substrate-dependent motility for host cell invasion and egress, which is dependent on an actomyosin motor complex called the glideosome. Apicomplexa have a small set of actin regulators, which are poorly conserved compared to their equivalents in higher eukaryotes. Actin-depolymerizing factors (ADFs) are key regulators responsible for accelerating actin turnover in eukaryotic cells. The activity of ADFs is regulated by membrane phosphoinositides. Malaria parasites express two ADF isoforms at different life stages. ADF1 differs substantially from canonical ADF/cofilins and from Plasmodium ADF2 in terms of both structure and function. Here, we studied the interaction of both Plasmodium ADFs with phosphoinositides using biochemical and biophysical methods and mapped their binding sites on ADF1. Both Plasmodium ADFs bind to different phosphoinositides, and binding in vitro requires the formation of vesicles or micelles. Interaction with phosphoinositides increases the α-helical content of the parasite ADFs, and the affinities are in the micromolar range. The binding site for phosphatidylinositol 4,5-bisphosphate in PfADF1 involves a small, positively charged surface patch.