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This study evaluated the effect of different potassium supplementation dosages on the physiological responses of Pangasianodon hypophthalmus reared in an aquaponic system with Spinacia oleracea L. for 60 days. The system comprised of a rectangular fish tank of 168 l capacity (water volume = 100 l) with nutrient film technique (NFT)–based hydroponic component with fish to plant ratio of 2.8 kg m−3: 28 plants m−2 in all the treatments. The osmoregulatory and stress parameters of P. hypophthalmus at four different potassium dosages of T1 (90 mg l−1), T2 (120 mg l−1), T3 (150 mg l−1) and T4 (180 mg l−1) were compared with C (control, 0 mg l−1) to examine the potassium level to be applied to aquaponics. The water quality parameters and fish production were found to have no adverse impact due to potassium supplementation. The spinach yield during two harvests, i.e., before and after potassium supplementation, revealed that the yield was significantly higher (P < 0.05) after supplementation with the highest yield in T3 and T4. The osmoregulatory parameters such as plasma osmolality, Na+, K+ ATPase activity in gill and plasma ionic profile (Cl−, Ca2+ and Na+) showed an insignificant variation (P > 0.05) between control and treatments except for higher plasma potassium concentration (1.98 ± 0.19 mmol l−1) in T4. The stress and antioxidant enzyme analysis exhibited significantly higher plasma glucose and superoxide dismutase (SOD) activity in gill and liver in T4, whereas cortisol and catalase showed an insignificant difference (P > 0.05). The experimental findings demonstrated that the potassium dosage up to 150 mg l−1 could be suggested as optimum for P. hypophthalmus and spinach aquaponics without impairing the health and oxidative status of P. hypophthalmus.