Macrophage migration inhibitory factor homologues have been identified from several genera of parasites, including Plasmodium, and have shown some functional similarities to the host molecule. It was hypothesized that MIF molecules can act as a regulator in host-parasite interaction in favor of parasites survival during malaria infection. Although there has been some progress in recent studies, the biological function of the malaria parasite-derived MIF is still far from clear. In this study, cDNA of Pfmif was synthesized from mRNA of Plasmodium falciparum 3D7 strain and the recombinant protein was generated and analyzed for both enzymatic and chemotactic activities. The Plasmodium-derived MIF homologue molecules are conservative both inter-strain and interspecies. And all the sequences of them have typical structure of CC chemokine family: CC-C-C. PfMIF was proved to have chemotactic activity on human monocytes, which was similar to human-derived MIF, but at lower concentration than the latter. Meanwhile, the proline at position 2 was confirmed to be important for its tautomerase activity. With specific monoclonal and polyclonal antibodies, we demonstrated the release of PfMIF from cultured parasite-infected erythrocytes and the secretion of it from transfected eukaryotic cells in vitro, and more importantly, we found the existence of parasite derived MIF homologue in the sera of the patients infected by P. falciparum. These results will contribute to the understanding of the parasite-derived MIFs role during malaria infection.