A 56-day study was performed to examine the effect of freshwater (FW) and brackish water (BW 6‰ salinity) on the antioxidant ability, Na+/K+-ATPase (NKA) activities, histology, and transcriptome of the gill and kidney tissue in juvenile silver carp (Hypophthalmichthys molitrix). The results show that when juvenile silver carp were exposed to 6‰ salinity, the activities of superoxide dismutase (SOD) and catalase (CAT) were shown to be substantially increased (p < 0.05), while glutathione peroxidase (GSH-PX) activities in gill were not significantly affected (p < 0.05). In kidney tissue, SOD, CAT, and GSH-PX, enzyme activities peaked at 24, 8, and 4 h, respectively, but were not significantly different compared with the control group (p < 0.05). In addition, significant effects of salinity were observed for the NKA level in both the gills and kidney tissues (p < 0.05). The gill filaments of juvenile silver carp under the BW group all underwent adverse changes within 72 h, such as cracks and ruptures in the main part of the gill filaments, bending of the gill lamellae and enlargement of the gaps, and an increase in the number of mucus and chloride-secreting cells. Transcriptome sequencing showed 171 and 261 genes in the gill and kidney tissues of juvenile silver carp compared to the BW group, respectively. Based on their gene ontology annotations, transcripts were sorted into four functional gene groups, each of which may play a role in salt tolerance. Systems involved in these processes include metabolism, signal transduction, immunoinflammatory response, and ion transport. The above findings indicate that the regulation processes in juvenile silver carp under brackish water conditions are complex and multifaceted. These processes and mechanisms shed light on the regulatory mechanism of silver carp osmolarity and provide a theoretical foundation for future research into silver carp growth in brackish water aquaculture area.