Effective clinical immunity that protects against symptomatic malaria in humans develops gradually after repeated exposure to Plasmodium falciparum. During intra-erythrocytic development, P. falciparum expresses novel antigens on the surface of infected erythrocytes, including PfEMP1, RIFIN, STEVOR, and SURFIN. Antibodies to surface antigens are typically variant-specific and appear to play an important role in contributing to protective immunity in humans. However, the significance of different surface antigens as targets of acquired immunity remains unclear. In our study, we used an innovative approach to evaluate the importance of surface antigens as antibody targets. This was achieved using genetically-modified P. falciparum with disrupted parasite protein trafficking, achieved by deletion of the skeletal-binding protein 1 (knock-out), and parasites with inhibited PfEMP1 expression (knock-down). Currently, only PfEMP1 is trafficked via the SBP1-pathway to the infected erythrocyte surface. We used high-throughput flow cytometry-based assays to measure antibody reactivity to the infected erythrocyte surface and opsonic phagocytosis assays using samples from cohort studies in Papua New Guinea and Kenya. Comparison between the parental and transgenic parasites allowed us to quantify the proportion of antibodies targeting specific antigens on the infected erythrocyte surface. We found very little antibody response to the transgenic parasites, suggesting that the SBP1 plays a central role in the trafficking of virulence proteins to the surface of infected erythrocytes. Our findings are consistent with PfEMP1 being the dominant target of acquired antibodies. These findings enhance our understanding of acquired immunity to human malaria and have significant implications for vaccine development.