To understand the underlying mechanism of the Pacific white shrimp Litopenaeus vannamei responding to acute salinity stress. RNA-seq was used to determine the transcriptome response of shrimp muscle and gill after ambient salinity changed from salinity of 20 (control) to 3 in 24 h. A total of 281.4 million reads were obtained and assembled into 105.153 contigs with an average length of 984 bp. Comparison of gene expression between shrimp exposed to salinity of 3 and the salinity control revealed that 991 and 3.709 genes were differently expressed in the gill and muscle, respectively. Both in muscle and gill, the changes of pathway can be categorized into oxidative pathways, signal transduction pathways, and metabolism pathways. More pathways significantly responded in gill than in muscle in metabolism and signal transduction. The significant change of pathways revealed that under acute low salinity stress, the increase of energy derived from carbohydrate, amino acid, or lipid in gill could satisfy the extra energy requirement of shrimp under salinity stress, but also lead to an overproduction of reactive oxygen species. For the maintenance of homeostasis, protein ubiquitination and relevant pathways were activated to remove the excessive reactive oxygen species and metabolite waste.