As a result of the manufacturing errors in the main parts of spindle, and the errors in assembly process of spindle, as well as the non-uniform heat distribution during spindle operation, the non-uniformly distributed preload in rolling bearing is inevitable. The non-uniform preload has not only axial force on the rolling bearing, but also the bending moment around the rolling bearing center. The effect of preload in bearing being non-uniform on the spindle performance is still unclear. In order to analyze the spindle performance with rolling bearing under non-uniform preload, a spindle test system with adjustable non-uniform distribution preload is built. By changing the magnitude of preload force on each action point, it can make the preload force distribute non-uniformly. In the static state of spindle, upon the change of non-uniformly distributed preload, the radial displacement of spindle shaft end is also changed, while in the dynamic state of spindle, it will lead to the change of the rotation center of spindle as a result of bending moment accompanying the non-uniform preload. Furthermore, the dynamic and static results show that when the preload is under non-uniform distribution, the spindle performance is significantly different from that under uniform preload. So this study would have certain reference value to check the spindle performance and explore novel preload method for rolling bearing.