[Problem] To provide a multi-layer culture vessel operation system that facilitates handling operations of a multi-layer culture vessel. [Solution] This multi-layer culture vessel operation system comprises: a carriage device 20 that is movable with a multi-layer culture vessel 30 mounted thereon; and an operation device 10 capable of holding and rotating the multi-layer culture vessel 30. The carriage device 20 has: a carriage 21 having wheels 22; and a fixing member 23 that is removably mounted on the carriage 21 and fixes the multi-layer culture vessel 30 to the carriage. The operation device 10 has: a rotation unit 11 that holds the multi-layer culture vessel 30 and performs a rotation operation to rotate the multi-layer culture vessel 30 about a first rotation axis and/or a second rotation axis; a shaking unit 13 that holds the multi-layer culture vessel 30 and performs a shaking operation to shake the multi-layer culture vessel 30 in the horizontal direction; and a control unit 15 that causes the shaking unit 13 to perform the shaking operation following the rotation operation by the rotation unit 11 without returning the multi-layer culture vessel 30 to the carriage 21.
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