The membrane asymmetry regulated by P4-ATPases in crucial for optimal functioning of eukaryotic cells. The underlying spatial translocation or flipping of specific lipids is usually assured by respective P4-ATPases coupled to conforming non-catalytic subunits. Our previous work has identified five P4-ATPases (TgP4-ATPase1-5) and three partner proteins (TgLem1-3) in the intracellular protozoan pathogen, Toxoplasma gondii. However, their flipping activity, physiological relevance and functional coupling remain unknown. Herein, we demonstrate that TgP4-ATPase1 and TgLem1 work together to translocate phosphatidylserine (PtdSer) during the lytic cycle of T. gondii. The two proteins localize in the plasma membrane at the invasive (apical) end of its acutely-infectious tachyzoites stage. The knockout of P4-ATPase1 as well as depletion of Lem1 severely disrupt the asexual reproduction of tachyzoites along with their ability of translocate PtdSer. Moreover, phenotypic analysis of individual mutants revealed their requirement for the motility, egress and invasion of tachyzoites. Our findings disclose the significance and mechanism of PtdSer flipping during the lytic cycle and emphasize P4-ATPase1/Lem1 heterocomplex as a potential drug target in T. gondii.