Sturgeons are euryhaline fish species that have developed specific mechanisms of osmotic and ion regulation to adapt to waters of varying salinity. For the aim to elucidate the osmoregulation strategy behind its high salinity tolerance of sturgeons, the transcriptomes of gills in Siberian sturgeon Acipenser baeri under salinity stress (30 ppt) were sequenced using deep-sequencing platform Illumina/HiSeq-2500 and differential expression genes (DEGs) were identified. A total of 167, 501, 278 clean reads were obtained and 280, 238 unigenes were composed of those clean reads with the mean length of 520nt, and the N50 of 630 bp. Unigenes Sequence alignment was implemented via KEGG, KOG, NT, NR, PFAM, Swiss-Prot, and GO databases. 62, 242 unigenes (22.21%) were annoated in at least one database. 11380 significantly differentially expressed unigenes were found, 6969 of which were up-regulated and 4411 were down-regulated by salinity stress. Amongst the top 20 KEGG pathways with the most amount of annotation sequences, some pathways such as glycerophospholipid metabolism, fatty-acid biosynthesis, glycolysis/gluconeogenesis, oxidative phosphorylation have been comprehensively proved to be relevant to osmoregulation. Despite of these, three possible osmoregulation-related signaling pathways as lipid metabolism related pathways, tight junction pathway and thyroid hormone signaling pathway have been widely analyzed in the current study. In all DEGs, some of the typical genes involved in osmoregulation, including calcium-transporting ATPase 4 (ATP2B4), Na+/K+-ATPase alpha subunit (α-NKA), potassium-transporting ATPase alpha chain 1 (ATP4A) and Ras GTPase-activating protein (RasGAP) etc were also identified. RNA-seq results were validated with quantitative real-time PCR (qPCR), the 12 selected genes showed a consistent direction in both DGE library and qPCR analysis, proving that the RNA-seq results are reliable. The present results would be helpful to elucidate the osmoregulation mechanism of aquatic animals adapting to salinity challenge.