Through the method of combining theoretical calculation and experimental research，the reasons and the influencing factors for the looseness of the support bolts of the Three Gorges ship lift tumble gate opening and closing cylinders are discussed，and the countermeasures are put forward. The results show that：1）Under the action of alternating load，the tumble gate is opened and closed frequently. The looseness of the bolts is caused by both material looseness resulted from plastic deformation of each contact surface of bolted connection of the opening and closing cylinder under large load and structural looseness caused by micro slip of screw pair.2）With the increase of the number of reciprocating opening and closing of tumble gate，the looseness of the bolts gradually increased until they failed.3）Through measures such as regular inspection，ultrasonic bolt pre-tightening force detection，and re-tightening，the looseness of the support bolts of the tumble gate opening and closing cylinder can be effectively reduced.