Litopenaeus vannamei is a typical euryhaline decapod model to study the osmoregulation mechanism in crustaceans. The proteomic was undertaken using isobaric tags for relative and absolute quantification together with the reverse phase in high-performance liquid chromatography mass spectrometry to quantitatively identify the proteins differentially expressed in the hepatopancreas under low salinity stress (3 psu) compared with the control salinity (25 psu). 533 proteins and 84 differentially expressed proteins were identified including 58 proteins with the 1.2-fold cut-off value under chronically low salinity stress. Among these proteins, 26 were up-regulated while 32 were down-regulated. 48 out of 58 differentially expressed proteins were annotated in the Uniprot database and were mapped into 38 pathways by KEGG analysis. These proteins were categorized into the pathways for energy metabolism, signaling, immunization and detoxification, lipid and protein metabolism. A more active glycometabolism, positive response detoxification pathway, immunosuppression and positive osmoregulation were identified in L. vannamei under low salinity stress. This study suggests that under chronically low salinity stress, L. vannamei showed low immunity and high demand for energy especially from glycometabolism. Signaling transfer related pathways, especially the Wnt signaling pathways were involved in the process of salinity adaption, but the in-depth mechanism warrants further investigation. Significance In this study, a comprehensive physiological response was studied using proteomics to reveal the underlying mechanism of adaptation to low salinity in L. vannamei, which was the first report on the proteomic response of crustacean to salinity stress. The extensive proteomic investigation on hepatopancreas under low salinity stress provides a new insight into the adaptive mechanism of this euryhaline crustacean species to low salinity.