As an important marine aquaculture species, the mud crab (Scylla paramamosain) is a good candidate for studying the osmoregulatory mechanism of crustaceans. While previous studies have focused on the osmoregulatory function of the gills, this study aims to explore the osmoregulatory function of the antennal glands. By the comparative transcriptomic analysis, we found the pathways of ion regulation including "proximal tubule bicarbonate reclamation" and "mineral absorption" were activated in the antennal glands of the crabs long-term dwelling in low salinity. The enhanced ionic reabsorption was associated with up-regulated ion transport genes such as NKA, CA-c, VPA, and NHE, and with energy metabolism genes such as MDH, SLC25, and PEPCK. The upregulation of NKA and CA-c was also verified by the increased enzyme activity. The lowered osmolality and ion concentration of the hemolymph and the enlarged labyrinth lumen and hemolymph capillary inside the antennal glands indicated the infiltration of external water and the responsively increase of urine excretion, which explained the requirement of enhanced ionic reabsorption. To further confirm these findings, we examined the change of gene expression, enzyme activity, internal ion concentration, and external ion concentration during a 96 h low salinity challenge with seven intervals. The results were basically consistent with the results as shown in the long-term low salinity adaptation. The present study provides valuable information on the osmoregulatory function of the antennal glands of S. paramamosain. The implication of this study in marine aquaculture is that it provides valuable information on the osmoregulatory mechanism of mud crabs, which can be used to improve their culture conditions and enhance their tolerance to salinity stress. The identified genes and pathways involved in osmoregulation can also be potential targets for genetic selection and breeding programs to develop more resilient mud crab strains for aquaculture.