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Macrophage migration inhibitory factor (MIF) is an evolutionarily ancient cytokine-like factor and plays a critical role in both innate and adaptive immunity. In the present study, two MIFs (designed as RpMIF-1 and RpMIF-2, respectively) were identified and characterized from the clam Ruditapes philippinarum by rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of RpMIF-1 and RpMFI-2 consisted of 531 and 722 nucleotides, encoding a polypeptide of 113 and 114 amino acid residues, respectively. Multiple alignments and phylogenetic analysis revealed that both RpMIF-1 and RpMIF-2 belonged to the MIF family. The conserved catalytic-site Pro2 for tautomerase activity was identified in the deduced amino acid sequences of RpMIFs. Both RpMIF-1 and RpMIF-2 transcripts were constitutively expressed in examined tissues of R. philippinarum with dominant expression in hepatopancreas, gills and hemocytes. Immunolocalization analysis showed that RpMIF-1 and RpMIF-2 proteins were expressed in examined tissues with the exception of adductor muscle and foot. After Vibrio anguillarum and Micrococcus luteus challenge, the mRNA expression of RpMIFs was significantly modulated in hemocytes, gills and hepatopancreas. Recombinant RpMIF-1 and RpMIF-2 proteins possessed significant tautomerase activity and oxidoreductase activity, indicating that these two proteins was perhaps involved in inflammatory responses. In summary, our results suggested that RpMIF-1 and RpMIF-2 played an important role in the innate immunity of R. philippinarum.Two MIFs were identified from Ruditapes philippinarum.RpMIFs were mainly expressed in hepatopancreas, gills and hemocytes.The mRNA expression levels of RpMIFs were modulated post bacterial challenge.rRpMIFs possessed tautomerase and oxidoreductase activity.