The effects of a range of different sublethal salinities were assessed on physiological processes and growth performance in the freshwater 'tra' catfish (Pangasianodon hypophthalmus) juveniles over an 8-week experiment. Fish were distributed randomly among 6 salinity treatments [2, 6, 10, 14 and 18 g/L of salinity and a control (0 g/L)] with a subsequent 13-day period of acclimation. Low salinity conditions from 2 to 10 g/L provided optimal conditions with high survival and good growth performance, while 0 g/L and salinities >14 g/L gave poorer survival rates (p < 0.05). Salinity levels from freshwater to 10 g/L did not have any negative effects on fish weight gain, daily weight gain, or specific growth rate. Food conversion ratio, however, was lowest in the control treatment (p < 0.05) and highest at the maximum salinities tested (18 g/L treatment). Cortisol levels were elevated in the 14 and 18 g/L treatments after 6 h and reached a peak after 24-h exposure, and this also led to increases in plasma glucose concentration. After 14 days, surviving fish in all treatments appeared to have acclimated to their respective conditions with cortisol levels remaining under 5 ng/mL with glucose concentrations stable. Tra catfish do not appear to be efficient osmoregulators when salinity levels exceed 10 g/L, and at raised salinity levels, growth performance is compromised. In general, results of this study confirm that providing culture environments in the Mekong River Basin do not exceed 10 g/L salinity and that cultured tra catfish can continue to perform well.