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Gel-enhanced liquid chromatography coupled with tandem mass spectrometry (GeLC-MS/MS) was used to analyze the proteome of Macrobrachium rosenbergii hepatopancreas responded to Poly (I:C). GeLC-MS/MS analysis identified 515 differentially-expressed proteins with ≥1.5 and ≤ −0.5 log2 fold change. Of these, 195 differentially-expressed proteins were significantly matched to known proteins in the database, of which 102 proteins were up-regulated and 93 proteins were down-regulated. These proteins were classified into 21 categories, i.e. metabolic process, oxidative stress response, signaling, transcription, translation, cell cycle, transport, etc. Several immune factors were up-regulated upon Poly (I:C) injection. Protein-protein interaction network analysis of these immune factors identified three major protein clusters including RNAi, stress responses, and Toll pathway-proPO system, implying that Poly (I:C) activates immune responses in prawn through several mechanisms.A total of 195 known proteins expressed differentially in response to Poly (I:C) and could be classified into 21 categories.Poly (I:C) stimulation increased the expression level of several immune factors in prawn.Three clusters of immune factors, including RNAi, stress responses, and Toll pathway-proPO system were identified.