ABSTRACTHere, we report that the inhibition of the PP2A subfamily by okadaic acid results in an accumulation of polysaccharides in the acute infection stage (tachyzoites) ofToxoplasma gondii, which is a protozoan of global zoonotic importance and a model for the apicomplexan parasites. The loss of the catalytic subunit α of PP2A (ΔPP2Acα) in RHΔku80leads to the polysaccharide accumulation phenotype in the base of tachyzoites as well as residual bodies and significantly compromises the intracellular growthin vitroand the virulencein vivo. A metabolomic analysis revealed that the accumulated polysaccharides in ΔPP2Acαare derived from interrupted glucose metabolism, which affects the production of ATP and energy homeostasis in theT. gondiiknockout. The assembly of the PP2Acα holoenzyme complex involved in the amylopectin metabolism in tachyzoites is possibly not regulated by LCMT1 or PME1, and this finding contributes to the identification of the regulatory B subunit (B′/PR61). The loss of B′/PR61 results in the accumulation of polysaccharide granules in the tachyzoites as well as reduced plaque formation ability, exactly the same as ΔPP2Acα. Taken together, we have identified a PP2Acα-B′/PR61 holoenzyme complex that plays a crucial role in the carbohydrate metabolism and viability inT. gondii, and its deficiency in function remarkably suppresses the growth and virulence of this important zoonotic parasite bothin vitroandin vivo. Hence, rendering the PP2Acα-B′/PR61 holoenzyme functionless should be a promising strategy for the intervention ofToxoplasmaacute infection and toxoplasmosis.IMPORTANCEToxoplasma gondiiswitches back and forth between acute and chronic infections, mainly in response to host immunologic status, which is characterized by flexible but specific energy metabolism. Polysaccharide granules are accumulated in the acute infection stage ofT. gondiithat have been exposed to a chemical inhibitor of the PP2A subfamily. The genetic depletion of the catalytic subunit α of PP2A leads to this phenotype and significantly affects the cell metabolism, energy production, and viability. Further, a regulatory B subunit PR61 is necessary for the PP2A holoenzyme to function in glucose metabolism and in the intracellular growth ofT. gondiitachyzoites. A deficiency of this PP2A holoenzyme complex (PP2Acα-B′/PR61) inT. gondiiknockouts results in the abnormal accumulation of polysaccharides and the disruption of energy metabolism, suppressing their growth and virulence. These findings provide novel insights into cell metabolism and identify a potential target for an intervention against aT. gondiiacute infection.