ABSTRACTCyclic invasion of red blood cells (RBCs) byPlasmodiummerozoites is associated with the symptoms and pathology of malaria. Merozoite invasion is powered actively and rapidly by a parasite actomyosin motor called the glideosome. The ability of the glideosome to generate force to support merozoite entry into the host RBCs is thought to rely on its stable anchoring within the inner membrane complex (IMC) through membrane-resident proteins, such as GAP50 and GAP40. Using a conditional knockdown (KD) approach, we determined that PfGAP40 was required for asexual blood-stage replication. PfGAP40 is not needed for merozoite egress from host RBCs or for the attachment of merozoites to new RBCs. PfGAP40 coprecipitates with PfGAP45 and PfGAP50. During merozoite invasion, PfGAP40 is associated strongly with stabilizing the expression levels of PfGAP45 and PfGAP50 in the schizont stage. Although PfGAP40 KD did not influence IMC integrity, it impaired the maturation of gametocytes. In addition, PfGAP40 is phosphorylated, and mutations that block phosphorylation of PfGAP40 at the C-terminal serine residues S370, S372, S376, S405, S409, S420, and S445 reduced merozoite invasion efficiency. Overall, our findings implicate PfGAP40 as an important regulator for the gliding activity of merozoites and suggest that phosphorylation is required for PfGAP40 function.IMPORTANCERed blood cell invasion is central to the pathogenesis of the malaria parasite, and the parasite proteins involved in this process are potential therapeutic targets. Gliding motility powers merozoite invasion and is driven by a unique molecular motor termed the glideosome. The glideosome is stably anchored to the parasite inner membrane complex (IMC) through membrane-resident proteins. In the present study, we demonstrate the importance of an IMC-resident glideosome component, PfGAP40, that plays a critical role in stabilizing the expression levels of glideosome components in the schizont stage. We determined that phosphorylation of PfGAP40 at C-terminal residues is required for efficient merozoite invasion.