Simple Summary: Procambarus clarkii is an economic freshwater aquaculture species which is popular with consumers for its delicious flavor and high protein content. Salinization of freshwater ecosystems is an increasingly pressing global issue that poses a significant threat to aquaculture. Salinity is an important environmental factor directly affecting the metabolism, growth, reproduction, and physiological processes of aquatic animals. In this study, crayfish were subjected to acute low salt (6 ppt) and high salt (18 ppt) stress and investigated using transcriptome sequencing technology. The response of the crayfish to different salinity stresses, especially immunity, metabolism, ion transport, and osmoregulation, was analyzed to illustrate the resistance mechanism of crayfish facing salt stress. The results of this study are intended to deepen our understanding of the mechanisms by which freshwater organisms respond to salinity stress and provide useful references for the healthy culture of crayfish and the utilization of saline soils. Salinization of freshwater ecosystems is a pressing global issue. Changes in salinity can exert severe pressure on aquatic animals and jeopardize their survival. Procambarus clarkii is a valuable freshwater aquaculture species that exhibits some degree of salinity tolerance, making it an excellent research model for freshwater aquaculture species facing salinity stress. In the present study, crayfish were exposed to acute low salt (6 ppt) and high salt (18 ppt) conditions. The organisms were continuously monitored at 6, 24, and 72 h using RNA-Seq to investigate the mechanisms of salt stress resistance. Transcriptome analysis revealed that the crayfish responded to salinity stress with numerous differentially expressed genes, and most of different expression genes was observed in high salinity group for 24h. GO and KEGG enrichment analyses indicated that metabolic pathways were the primary response pathways in crayfish under salinity stress. This suggests that crayfish may use metabolic pathways to compensate for energy loss caused by osmotic stress. Furthermore, gene expression analysis revealed the differential expression of immune and antioxidant-related pathway genes under salinity stress, implying that salinity stress induces immune disorders in crayfish. More genes related to cell proliferation, differentiation, and apoptosis, such as the Foxo, Wnt, Hippo, and Notch signaling pathways, responded to high-salinity stress. This suggests that regulating the cellular replication cycle and accelerating apoptosis may be necessary for crayfish to cope with high-salinity stress. Additionally, we identified 36 solute carrier family (SLC) genes related to ion transport, depicting possible ion exchange mechanisms in crayfish under salinity stress. These findings aimed to establish a foundation for understanding crustacean responses to salinity stress and their osmoregulatory mechanisms.