The disease-causing blood-stage of the Plasmodium falciparum lifecycle begins with invasion of human erythrocytes by merozoites. Many vaccine candidates with key roles in binding to the erythrocyte surface and entry are secreted from the large bulb-like rhoptry organelles at the apical tip of the merozoite. Here we utilise high- and super-resolution imaging and identify an essential role for the conserved protein P. falciparum Cytosolically Exposed Rhoptry Leaflet Interacting protein 1 (PfCERLI1) in rhoptry function. Using biochemical techniques and quantitative super-resolution microscopy, we show that PfCERLI1 localises to the cytosolic face of the rhoptry bulb membrane and knockdown of PfCERLI1 inhibits merozoite invasion. While schizogony and merozoite organelle biogenesis appear normal, reduction in PfCERLI1 expression appears to block secretion of key rhoptry antigens that coordinate merozoite invasion. While further studies need to be undertaken to determine the fine detail of how PfCERLI1 knockdown causes these changes in rhoptry function, identification of PfCERLI1’s direct association with release of rhoptry antigens is a key step in understanding the complex molecular events that control rhoptry secretion during invasion. This study makes extensive use of semi-automated quantitative immunofluorescence microscopy and highlights how this powerful tool can be used to study the process of invasion.