A 90-day experiment was conducted to study the impact of salinity on growth and physiological response of Pangasionodon hypophthalmus fingerlings with different saline conditions such as 0 (control), 5 (T1), 10 (T2) and 15 (T3) ppt. Fingerling with an average body weight of 8.47 g, stocked uniformly to all the treatments with a stocking density of 40 fish/tank in three replicates and fed with a commercial feed (crude protein 30%). At fortnight interval, 25% (10 fish/tank) of the stocks collected to record the length and weight of individual fish. Blood and liver samples collected at 30-day interval for assessing the stress, digestive enzymes and immunological parameters. The average body weight and specific growth rate were higher in 0 and 5 ppt reared fish compared to those at higher salinities. The best FCR (1.59 ± 0.02), higher FER (0.63 ± 0.01) and PER (1.03 ± 0.01) were observed in 0 ppt group. No significant difference was observed in the survival rate of fish among the treatments except 15 ppt group. The RNA:DNA ratio of muscle was significantly higher at highest salinity (15 ppt) which indicated the higher osmotic energy demand at higher salinities. The immune parameters such as respiratory burst activity, superoxide dismutase and myeloperoxidase were significantly higher in 0, 5 and 10 ppt groups. Significantly, lower catalase and lysozyme activity were observed in 0 ppt group. The fishes indicated stress response in terms of increased cortisol, glucose, aspartate aminotransferase and alanine aminotransferase at higher salinities. The reduced digestive enzymes of amylase and protease activity were also observed in 15 ppt raised fishes which can be further correlated with their reduced FER and PER. Hence, the present study concludes that P. hypophthalmus reasonably exhibited good growth performance, immune, digestion and stress mitigation ability up to 10 ppt which indicates that Pangasius culture is possible in brackish water (up to 10 ppt) without any negative effect on its homeostasis.