Melanoma differentiation-associated gene 5 (MDA5) belongs to RIG-I like receptor (RLR) family, which detects cytosolic viral RNA component in immune response. In this study, MDA5 orthologue of black carp (Mylopharyngodon piceus) has been cloned and characterized. The full-length cDNA of black carp MDA5 (bcMDA5) comprises 3244 nucleotides and the predicted bcMDA5 protein contains 984 amino acids. The constitutive transcription of bcMDA5 was extremely low in all the tested tissues, which included gill, skin, muscle, intestine, kidney, spleen, liver and heart. However, bcMDA5 mRNA level was much enhanced in most selected tissues in response to GCRV or SVCV infection. bcMDA5 migrated around 120 KDa in immunoblot and was identified as a cytosolic protein by immunofluorescent staining in both EPC and HeLa cells. Expressing bcMDA5 in EPC cells resulted in the induction of promoter activity of zebrafish IFN3 or fathead minnow IFN. The EPC cells expressing bcMDA5 obtained improved antiviral ability against both SVCV and GCRV. When EPC cells were co-transfected with plasmids expressing bcMDA5 and bcLGP2, the induced IFN expression by bcMDA5 was obviously enhanced. EPC cells expressing both bcMDA5 and bcLGP2 owned much improved antiviral ability than those cells expressing only bcMDA5 or bcLGP2. In general, our data support the conclusion that bcMDA5 plays an important role in the antiviral innate immune response of black carp and bcLGP2 acts as a positive regulator in bcMDA5 mediated signaling.