In the present study, juvenile striped catfish (Pangasianodon hypophthalmus), a freshwater fish species, have been chronically exposed to a salinity gradient from freshwater to 20 psu (practical salinity unit) and were sampled at the beginning (D20) and the end (D34) of exposure. The results revealed that the intestinal microbial profile of striped catfish reared in freshwater conditions were dominated by the phyla Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia. Alpha diversity measures (observed OTUs (operational taxonomic units), Shannon and Faith’s PD (phylogenetic diversity)) showed a decreasing pattern as the salinities increased, except for the phylogenetic diversity at D34, which was showing an opposite trend. Furthermore, the beta diversity between groups was significantly different. Vibrio and Akkermansia genera were affected differentially with increasing salinity, the former being increased while the latter was decreased. The genus Sulfurospirillium was found predominantly in fish submitted to salinity treatments. Regarding the host response, the fish intestine likely contributed to osmoregulation by modifying the expression of osmoregulatory genes such as nka1a, nka1b, slc12a1, slc12a2, cftr, and aqp1, especially in fish exposed to 15 and 20 psu. The expression of heat shock proteins (hsp) hsp60, hsp70, and hsp90 was significantly increased in fish reared in 15 and 20 psu. On the other hand, the expression of pattern recognition receptors (PRRs) were inhibited in fish exposed to 20 psu at D20. In conclusion, the fish intestinal microbiota was significantly disrupted in salinities higher than 10 psu and these effects were proportional to the exposure time. In addition, the modifications of intestinal gene expression related to ion exchange and stressful responses may help the fish to adapt hyperosmotic environment.