A multi-layer culture vessel operation system comprising: a cart device movable with a multi-layer culture vessel including a plurality of trays mounted thereon; and an operational device capable of holding and rotating the multi-layer culture vessel, wherein the cart device includes: a cart having wheels; and a fixing part that is removably mounted on the cart, and fixes the multi-layer culture vessel to the cart, wherein the operational device includes: a rotating portion that holds the multi-layer culture vessel together with the fixing part, and performs a rotation operation of rotating the multi-layer culture vessel around at least one of a first rotation axis and a second rotation axis; a shaking portion that holds the multi-layer culture vessel together with the fixing part, and performs a shaking operation of shaking the multi-layer culture vessel in a horizontal direction; and a control portion that controls operations of the rotating portion and the shaking portion, and wherein the control portion causes the shaking portion to perform the shaking operation following the rotation operation by the rotating portion, without returning the multi-layer culture vessel to the cart. a cart device movable with a multi-layer culture vessel including a plurality of trays mounted thereon; and an operational device capable of holding and rotating the multi-layer culture vessel, wherein the cart device includes: a cart having wheels; and a fixing part that is removably mounted on the cart, and fixes the multi-layer culture vessel to the cart, wherein the operational device includes: a rotating portion that holds the multi-layer culture vessel together with the fixing part, and performs a rotation operation of rotating the multi-layer culture vessel around at least one of a first rotation axis and a second rotation axis; a shaking portion that holds the multi-layer culture vessel together with the fixing part, and performs a shaking operation of shaking the multi-layer culture vessel in a horizontal direction; and a control portion that controls operations of the rotating portion and the shaking portion, and wherein the control portion causes the shaking portion to perform the shaking operation following the rotation operation by the rotating portion, without returning the multi-layer culture vessel to the cart. a cart having wheels; and a fixing part that is removably mounted on the cart, and fixes the multi-layer culture vessel to the cart, wherein the operational device includes: a rotating portion that holds the multi-layer culture vessel together with the fixing part, and performs a rotation operation of rotating the multi-layer culture vessel around at least one of a first rotation axis and a second rotation axis; a shaking portion that holds the multi-layer culture vessel together with the fixing part, and performs a shaking operation of shaking the multi-layer culture vessel in a horizontal direction; and a control portion that controls operations of the rotating portion and the shaking portion, and wherein the control portion causes the shaking portion to perform the shaking operation following the rotation operation by the rotating portion, without returning the multi-layer culture vessel to the cart. a rotating portion that holds the multi-layer culture vessel together with the fixing part, and performs a rotation operation of rotating the multi-layer culture vessel around at least one of a first rotation axis and a second rotation axis; a shaking portion that holds the multi-layer culture vessel together with the fixing part, and performs a shaking operation of shaking the multi-layer culture vessel in a horizontal direction; and a control portion that controls operations of the rotating portion and the shaking portion, and wherein the control portion causes the shaking portion to perform the shaking operation following the rotation operation by the rotating portion, without returning the multi-layer culture vessel to the cart.