Abstract: The giant freshwater prawn (GFP) Macrobrachium rosenbergii is one of the important economically freshwater shrimp species, and its annual production in China is dominant in the world. However, its tolerance to low temperatures is extremely poor, and acute low temperatures can lead to large-scale deaths and cause huge economic losses. In order to explore genes related to the GFP response to acute low temperature, comparative transcriptomic analyses were performed for the hepatopancreas of adult GFPs exposed to low temperature. The low temperature stress group (16℃) and the control group (24℃) were set up. The water temperature of the stress group was decreased from 24℃ to 16℃ at a rate of around 2℃/h through adding ice cubes. The hepatopancreas were collected for transcriptomic analyses from exposed shrimps respectively at 1h, 3h, 6h, 12h, 24h after acute cooling at 16℃, rewarming to 24℃ and control group. The results of differentially expressed genes (DEGs) showed that 1702 DEGs were identified between samples of cooling for 1h and the control group (M1 vs. C0); the number of DEGs between the stress group and the control group began to increase gradually with stress time, and reached the maximum at 6h (M3 vs. C0, a total of 2899), then gradually decreased, maintaining the homeostasis under low temperature stress. After rewarming to 24℃, the number of DEGs (M6 vs. C0, 1969) returned to the level of 1h stress. Additionally, the DEGs number (5062) between samples of stress for 3h and rewarming to 24℃ (M2 vs. M6) was almost 1.5 times of that (3516) between samples of stress for 1h and rewarming (M1 vs. M6). With the extension of time, the number of DEGs decreased gradually, suggesting that the homeostasis of the GFP had drastic changes in the first 3h of acute low temperature stress, but the adaptability to low temperature gradually increased with stress time, and a steady state under low temperature was established probably after being stressed from 3h to 6h, and after rewarming, the homeostasis returned to the equilibrium of short-term cold stress. KEGG enrichment analysis showed that DEGs was enriched in lysosome, starch and sucrose metabolism, antigen processing and presentation. Adhesion spots, ECM-receptor interaction, metabolism of cytochrome P450 to isobiotic substances, glutathione metabolism, oxidative phosphorylation, and p53 signaling pathway were also involved in the regulation of acute cold stress in the GFP. The common DEGs among all groups were clustered as the cell function and immunity modules and energy metabolism modules. In addition, the up-regulated cytochrome P450 2L1-like gene in the arachidonic acid metabolic signaling pathway was screened, and its expression increased firstly and then decreased with the increase of cold stress time. NADP-specific isocitrate dehydrogenase, an upregulated gene in glutathione metabolic signaling pathway, was firstly reduced and then increased with time. Expression of hexokinase, a down-regulated gene in starch and sucrose metabolic signaling pathways, increased and then decreased over time. The above mentioned enriched pathways indicated that the metabolic function of the GFP might have been seriously affected under acute low temperature stress, with a large amount of reactive oxygen species being produced, the balance of energy circulation being disrupted, and the immune system being also damaged, which was corroborated with the phenomena of fasting, slow movement, susceptibility to diseases and even death of the GFP under acute low temperature. Consequently, these screened pathways and genes may play important roles in energy metabolism and immune regulation during acute cold stress in the GFP. The present study provides basic data for revealing the molecular regulatory mechanism of the response of the GFP to acute low temperature stress, also provides a theoretical basis for the selective breeding of new cold-tolerant GFP varieties.