The malaria parasite (Plasmodium falciparum) possesses a plastid-derived, essential organelle called the apicoplast, which contains a redox system comprising plant-type ferredoxin (Fd) and Fd-NADP+ reductase (FNR). This system supplies reducing power for the crucial metabolic pathways in this organelle. Electron transfer between P. falciparum Fd (PfFd) and FNR (PfFNR) is performed with higher affinity and specificity than that of plant Fd and FNR. To investigate the mechanism for such superior protein-protein interaction, we searched for the Fd interaction sites on the surface of PfFNR. Basic amino acid residues on the FAD binding side of PfFNR were comprehensively substituted to acidic amino acids by site-directed mutagenesis. Kinetic analysis of electron transfer to PfFd and plant Fds, physical binding to immobilized PfFd and thermodynamics of the PfFd binding using these PfFNR mutants revealed that several basic amino acid residues including those in Plasmodium-specific insertion region are important for the interaction with PfFd. Majority of these basic residues are Plasmodium-specific and not conserved among plant and cyanobacteria FNRs. These results suggest that the interaction mode of Fd and FNR is diverged during evolution so that PfFd: PfFNR interaction meets the physiological requirement in the cells of Plasmodium species.