Temperature is an essential environmental factor affecting the viability of organisms. Temperature changes could cause damages or even death. The oriental river prawn, Macrobrachium nipponense, is widely distributed in East Asian countries, and has been an important economical aquaculture species in China since 1990s. This prawn belongs to crustaceans, which is a kind of ectotherms and very sensitive to temperature changes due to their relatively low internal metabolic heat production and their high thermal conductivity. In order to reveal the thermal adaptation mechanisms, M. nipponense were acclimated at 25 °C for 24 h, and successively challenged at 30 °C and 35 °C for 24 h in the same tank, respectively. And histological, biochemical and transcriptomic analyses were carried out on the gills and hepatopancreas under different temperatures. The results showed that different degrees of histological damages were discovered both in the gills and hepatopancreas of M. nipponense at 30 °C and 35 °C. The digestive, metabolic, and antioxidative capacity were enhanced in the gills and hepatopancreas of M. nipponense at 30 °C and 35 °C. A total of 2293 and 3304 differentially expressed genes (DEGs) were identified from the gills and hepatopancreas, respectively. These DEGs were significantly involved in the regulation of transcription, proteolysis, nucleus, cytoplasm, metal ion binding, and ATP binding by Gene ontology (GO) enrichment analysis. Furthermore, the DEGs in the hepatopancreas were significantly enriched in KEGG pathways including neuroactive ligand-receptor interaction, thyroid hormone synthesis, and ECM-receptor interaction. And the DEGs in the gills were enriched in KEGG pathways including cGMP-PKG signaling pathway, ribosome, and calcium signaling pathway. These results could be helpful for further understanding the molecular mechanisms underlying the thermal response of M. nipponense, and increasing knowledge of the regulation mechanisms of freshwater crustaceans in the context of global warming.