Plasmodium falciparum parasites in the merozoite stage invade human erythrocytes and cause malaria. Invasion requires multiple interactions between merozoite ligands and erythrocyte receptors. P. falciparum reticulocyte binding homolog 5 (PfRh5) forms a complex with the PfRh5-interacting protein (PfRipr) and Cysteine-rich protective antigen (CyRPA) and binds erythrocytes via the host receptor basigin. However, the specific role that PfRipr and CyRPA play during invasion is unclear. Using P. falciparum lines conditionally expressing PfRipr and CyRPA, we show that loss of PfRipr or CyRPA function blocks growth due to the inability of merozoites to invade erythrocytes. Super-resolution microscopy revealed that PfRipr, CyRPA, and PfRh5 colocalize at the junction between merozoites and erythrocytes during invasion. PfRipr, CyRPA, and PfRipr/CyRPA/PfRh5-basigin complex is required for triggering the Ca2+ release and establishing the tight junction. Together, these results establish that the PfRh5/PfRipr/CyRPA complex is essential in the sequential molecular events leading to parasite invasion of human erythrocytes. Display omitted •P. falciparum PfRh5-binding partners PfRipr and CyRPA are essential for merozoite invasion•PfRipr and CyRPA are linked to release of calcium into the erythrocyte•PfRh5/PfRipr/CyRPA complex binding to host receptor basigin is required for Ca2+ release•PfRh5/PfRipr/CyRPA complex forms at the interface between the merozoite and erythrocyte Plasmodium falciparum PfRipr and CyRPA form a complex with PfRh5, which mediates erythrocyte invasion. The specific role of PfRipr and CyRPA is unclear. Volz et al. (2016) show that PfRipr and CyRPA are essential for invasion and required for Ca2+ release during the sequential molecular events for invasion of erythrocytes.