The Na⁺/H⁺ antiporter in vacuolar membranes transports Na⁺ from the cytoplasm to vacuoles using a pH gradient generated by proton pumps, which could reduce Na⁺ toxicity. It is uncertain that whether the woody plants have the same mechanism. Through differential centrifugation and sucrose density gradient centrifugation, tonoplast vesicles were isolated from Populus tremula calli broken by blender. After establishing pH gradient by V-ATPase, Na⁺ could dissipate the pH gradient, which indicates that there is Na⁺/H⁺ antiporter in the tonoplast vesicles from Populus tremula calli (K_m=11.4 mmol/L). Amiloride could inhibit the Na⁺/H⁺ antiporter activity. The antiporter could transport Na⁺ and K⁺, the affinity for Na⁺ is higher. Salt stress decreased K_m and V_max.