Innate immunity is the primary defense of crustaceans against pathogens. Crustins, as antimicrobial peptides, are important to crustacean innate immunity. In this study, two kinds of Gly-rich crustin genes were cloned from Macrobrachium nipponense and were referred to as Mn-Gly-Cru1 and Mn-Gly-Cru2. These crustins belong to type II crustins with typical type II crustin structures. The full-length cDNA of Mn-Gly-Cru1 is 677 bp and contains a 576 bp open reading frame (ORF) encoding 191 amino acids. The full-length cDNA of Mn-Gly-Cru2 is 727 bp, with 573 bp ORF encoding 190 amino acids. The constructed phylogenetic tree indicated that Mn-Gly-Cru1 and Mn-Gly-Cru2 belong to the type IIa subfamily. RT-PCR analysis showed that Mn-Gly-Cru1 and Mn-Gly-Cru2 are widely distributed in various tissues. qRT-PCR results indicated that Mn-Gly-Cru1 is mainly expressed in the gills, whereas Mn-Gly-Cru2 is expressed at the highest level in hemocytes. The transcripts of Mn-Gly-Cru1 and Mn-Gly-Cru2 respond to bacterial or white spot syndrome virus (WSSV) stimuli. After injection of 48 h dsMnRelish, the expression of MnRelish, Mn-Gly-Cru1, and Mn-Gly-Cru2 were all inhibited. After WSSV, Vibrio parahaemolyticus, or Staphylococcus aureus challenge, MnRelish, Mn-Gly-Cru1, and Mn-Gly-Cru2 were all upregulated. However, the expression levels of MnRelish, Mn-Gly-Cru1, and Mn-Gly-Cru2 at 6 h bacteria or 36 h WSSV challenge were downregulated in Relish-silenced prawns when compared with the control (bacteria or WSSV challenge only, bacteria or WSSV challenge plus dsGFP injection). Results suggest that Mn-Gly-Cru1 and Mn-Gly-Cru2 play essential roles in M. nipponense innate immunity against bacteria or WSSV, and the expression levels of both genes are regulated by Relish transcriptional factor.