Bearing preload has a direct influence on the static and dynamic characteristics of rolling bearing-rotor systems. Considering the effect of centrifugal force and gyro moment, the dynamic model of 5 degrees of freedom bearing-rotor system was established in Romax software, and the influence of preload on bearing stiffness,working contact angle, and working life was studied. Additionally, an experimental test bench of the bearingrotor system was built to evaluate the bearing stiffness, working contact angle, working life, static deformation by static load and vibration response of the spindle system by dynamic unbalanced load, under different bearing preloads. As a result, the relationship curves between the preload and these parameters were obtained. Finally, the effect of preload on natural frequencies of the bearing-rotor system was studied, and the result shows that increasing preload is helpful to increase the natural frequencies of the bearing-rotor system.