Differentially expressed genes were identified. Their study revealed a few important salinity acclimation pathways, which may be helpful in understanding the molecular basis of osmoregulation and salinity adaptation in the crab. Even though cDNA microarray technology is a powerful tool for studying genome-wide gene expression, this technology fails to detect sequence variation and to recognize new genes or transcripts and can only be designed from limited expressed sequence tag data as the genome of Diperodon Portunus trituberculatus has not yet been determined. To date, there are 13,985 ESTs available for the crab in the Genebank, however, it remains insufficient for the comprehensive understanding of Portunus trituberculatus transcriptome. Many low expression transcripts would be missed from current EST data, which makes it difficult for further analysis on transcriptome. Next-generation high-throughput RNA sequencing technology is a recently-developed method for discovering, profiling, and quantifying RNA transcripts with several advantages over other expression profiling technologies including higher sensitivity and the ability to detect splicing isoforms and somatic mutations. Because it is not restricted by the unavailability of a genome reference sequence, this approach has been applied in decoding the genomes of several non-model organisms, providing valuable information in the understanding of gene function, cell responses and evolution. Significant progress has also been made in understanding the transcript expression of various marine crustacea by RNA-seq over the last two years, such as Litopenaeus vannamei, Fenneropenaeus chinensis, Eriocheir sinensis and Macrobrachium nipponense.The countable, almost digital, nature of RNA-seq data makes them particularly attractive for the quantitative analysis of transcript expression levels, which can give reliable measurements of transcript levels in one or more conditions. However, such investigations in Portunus trituberculatus have not been reported. In the present study, we examined the whole transcriptome responses to salinity stress of the Portunus trituberculatus for the first time using the Illumina’s sequencing technology. Considering individual monitoring of the Portunus trituberculatus responses to salinity stress, nine libraries were established from the gills of Portunus trituberculatus that exposed to optimal, low and high salinity seawater, Pimozide respectively. The study aimed to compare the expression patterns of the three conditions to better understand the transcriptomic regulation in Portunus trituberculatus to salinity stress and identify genes involved in osmoregulation of the crab. The results of this study are an important resource for future researches on mechanism of osmoregulation for marine invertebrates. In recent years, the next generation sequencing methods have also been applied to analyze transcriptomes of crustaceans using Illumina sequencing. Comparing with the traditional methods, the next-generation high-throughput DNA sequencing techniques provide more ideal methods for transcriptome analyses with high efficiency, low cost and high data output. The former studies on Portunus trituberculatus transcriptome were performed using traditional cDNA library and Sanger sequencing methods with RNA from many organs such as gill, eyestalk, blood and hepatopancreas, however, it remains insufficient for the comprehensive understanding of Portunus trituberculatus transcriptome. In our study, we generated 141,339 contigs of Portunus trituberculatus transcriptome based on the next generation sequencing techniques. Further assembly analysis showed that all contigs contributed to 94,511 unigenes.