A separation column connecting device includes: a column holder for retaining a separation column; a first fitting holder carrying a first fitting which includes a seal portion to be connected to an upstream seal portion of the separation column and connected with an upstream pipe; a second fitting holder carrying a second fitting which includes a seal portion to be connected to a downstream seal portion of the separation column and connected with a downstream pipe; a body member to which either one of the first fitting holder and the second fitting holder is fixed; a driver for moving, relative to the body member, the first fitting holder or the second fitting holder not fixed to the body member; a guide for guiding the column holder in a direction of movement driven by the driver; and an elastic body disposed between the column holder and the second fitting holder.