The invention provides an axial pre-tightening device of a ball screw. The device comprises a machine tool body, the front end of the machine tool body is provided with a servo motor bearing base, the rear end of the machine tool body is provided with a rear bearing base, a ball screw pair, a pressing nut, a bearing gland and a bearing assembly are arranged in the servo motor bearing base, a protruding guide tenon is arranged on the servo motor bearing base, a guide groove is formed in the position, corresponding to the servo motor bearing base, of the machine tool body, the guide tenon is matched with the guide groove, an adjusting screw rod is arranged in the guide tenon, the adjusting screw rod is connected with a fixing plate on the machine tool body, a boss is arranged on the position, corresponding to the rear bearing base, of the machine tool body, and the boss is in contact with the rear bearing base. After clearance fit of bearings, by means of displacement adjustment of the bearing bases, axial stretching and pre-tightening of the ball screw of a machine tool transmission system are achieved, adjustment precision is improved, axial force is increased, linear motion precision and dynamic response precision of the ball screw are greatly improved, and operation in assembly is facilitated.