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The mitogen-activated protein kinase kinase 4 (MKK4) is a key component of the c-Jun N-terminal kinase (JNK) signaling pathway and regulates multiple cellular activities. However, little is known about the roles of this kinase in pearl oyster. In this study, we identified an MKK4 homologue in Pinctada fucata by using a transcriptome database. Sequence analysis and protein structure prediction showed that PfMKK4 is highly conserved to MKK4 from other vertebrate and invertebrate species. Phylogenetic analysis revealed that PfMKK4 has the closest relationship with that from Crassostrea gigas. QPCR was used to investigate expression profiles in different healthy adult tissues and developmental stages of P. fucata. We found that PfMKK4 was ubiquitously expressed in all tissues and developmental stages examined except for in D-shaped larvae. Gene expression analysis suggested that PfMKK4 is involved in the response to the nucleus insertion operation. Lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid [poly(I:C)] stimulation in vivo reduced PfMKK4 mRNA expression at 6h, 48h and 48h, 72h, respectively. LPS and poly(I:C) induced PfMKK4 phosphorylation in a primary mantle cell culture. These results contribute to better understanding of the potential role played by PfMKK4 in protecting the pearl oyster from injury caused by grafting or disease.PfMKK4 exhibits all the typical features of MKK4 gene.PfMKK4 wide distributes in tissues and developmental stages.PfMKK4 gene was expressed throughout the whole developmental stages, while the highest level was detected in the polar body stage.PfMKK4 may be involved in post-graft processes.PfMKK4 is involved in immune response in Pinctada fucata.