The oriental river prawn Macrobrachium nipponense is an important aquaculture species in China, Vietnam, and Japan. This species could survive in the salinity ranging from 7 to 20 ppt and accelerate growth in the salinity of 7 ppt. To identify the genes and pathways in response to acute high salinity stress, M. nipponense was exposed to the acute high salinity of 25 ppt. Total RNA from hepatopancreas, gills, and muscle tissues was isolated and then sequenced using high-throughput sequencing method. Differentially expressed genes (DGEs) were identified, and a total of 632, 836, and 1246 DEGs with a cutoff of significant twofold change were differentially expressed in the hepatopancreas, gills, and muscle tissues, respectively. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome pathway enrichment analyses were conducted. These DEGs were involved in the GO terms of cellular process, metabolic process, membrane, organelle, binding, and catalytic activity. The DEGs of hepatopancreas and gill tissues were mainly enriched in PPAR signaling pathway, longevity regulating pathway, protein digestion and absorption, and the DEGs of muscle tissue in arginine biosynthesis, adrenergic signaling in cardiomyocytes, cardiac muscle contraction, and cGMP-PKG signaling pathway. Real-time PCR conducted with fifteen selected DEGs indicated high reliability of digital analysis using RNA-Seq. The results indicated that the M. nipponense may regulate essential mechanisms such as metabolism, oxidative stress, and ion exchange to adapt the alternation of environment, when exposed to acute high salinity stress. This work reveals the numbers of genes modified by salinity stress and some important pathways, which could provide a comprehensive insight into the molecular responses to high salinity stress in M. nipponense and further boost the understanding of the potential molecular mechanisms of adaptation to salinity stress for euryhaline crustaceans.