Actin polymerization and actin-myosin coupling activity most likely provide the driving force that the protozoan parasite Toxoplasma gondii has to exert to propulse itself during gliding and host cell entry. Nevertheless, little information is available on T. gondii tachyzoite actin dynamics, and in particular, the presence of actin filaments remains largely uncharacterized. Here, we report that the marine sponge peptide jasplakinolide, known to bind to filamentous actin, does indeed stabilize a pool of a parasite detergent-insoluble actin. This pool is likely to be formed by a dynamic assembled actin complex: first, it is competent for assembly/disassembly and secondly, it is sensitive to nucleotide phosphate concentration. In addition, T. gondii tachyzoites contain molecules which inhibit actin assembly and destabilize actin filaments. Thus, these activities could account for the remarkably low amount of the myosin-containing F-actin pool we describe here. Furthermore, when parasites are treated with cell-permeant jasplakinolide, they display a significant loss of both motility and host cell invasiveness. These data suggest that in vivo, the detergent-insoluble pool of actin is dynamic.