There was no significant difference in PfRh4 antibodies according to gender. Furthermore, Tulathromycin B adjustments for gender in multivariate models did not significantly alter model outputs; therefore, gender was not included in the final model. Recombinant protein constructs PfRh4.1, PfRh4.2, and PfRh4.4 were GST-fusion proteins; reactivity to GST was very low, and was not associated with risk of malaria, and deducting GST reactivity from the reactivity to Rh4-GST fusion proteins did not affect analysis outcomes. Reporting of study outcomes followed the MIOS guidelines for malaria immuno-epidemiology observational studies. These findings represent a significant advance towards identifying targets of human immunity to malaria. Our broad evaluation of PfRh4 combining prospective studies of associations between antibodies and protective immunity, the functional activity of human antibodies, and PfRh4 expression by P. falciparum in the population provide important evidence, for the first time, suggesting that PfRh4 is a target of human immunity. IgG1 and IgG3 were the predominant subclasses for antibodies to PfRh4, with very little IgG2 or IgG4, as observed for other merozoite antigens. High levels of IgG3 to PfRh4.9, containing the erythrocyte-binding region, were very strongly associated with reduced malaria risk; this is the strongest association with Orbifloxacin protection against malaria that we have observed in this cohort. Importantly, this protective association remained strong and significant after adjusting for potential confounding factors of age, residential location, and parasitemia status at enrolment. High levels of IgG3 to PfRh4.2 were also strongly associated with protection against clinical malaria and high-density parasitemia, but this association was not as strong as that seen for IgG3 to PfRh4.9. Interestingly, IgG1 to PfRh4.9 was not significantly associated with protective immunity, suggesting that IgG3 might be functionally more important. Although a detailed knowledge of the functions of IgG subclasses in humans is lacking, prior studies report important differences in the function of IgG1 and IgG3 in other systems, and known structural differences between IgG1 and IgG3 may influence specificity and function. Animal studies indicate that different IgG subclasses have different immunologic activities and roles, and lead to different outcomes. Further studies are needed to understand the functional differences between IgG1 and IgG3 to PfRh4 and other merozoite antigens. The association between high levels of antibodies and protection from high-density parasitemia, but not re-infection per se, is consistent with PfRh4 antibodies acting by inhibition of erythrocyte invasion and parasite replication, which we observed in vitro. Human immunity to malaria is almost certainly mediated by responses to multiple target antigens, but few of these target antigens have been identified or studied in detail. Data presented here provide important evidence that antibodies to PfRh4 contribute to the protective immune response in humans. We have previously reported that antibodies to the EBAs and PfRh2 are strongly associated with protection; collectively, these studies identify the EBA and PfRh invasion ligand families as important targets of immune responses in humans that appear to contribute to protection against malaria. Given that P. falciparum can vary the use and expression of EBA and PfRh4 invasion ligands as a possible means of immune evasion, it is likely that functional antibodies to multiple invasion ligands would be needed to provide effective immunity. Demonstrating the functional significance of PfRh4 antibodies, affinity-purified human antibodies against the PfRh4.9 protein, which contains the erythrocyte-binding region, were potent inhibitors of SA-independent invasion, which requires the PfRh4 ligand.