The machine tool joint is a very important factor in the overall machine tool dynamic analysis, and it has great effects on the machining performance of a machine tool. As a very important operation parameter, preload greatly influences the stiffness and the damping of a machine tool joint. This paper presents the effect of preload on the dynamic stiffness of the spindle nose of a horizontal machining center. By discussing types and distribution of machine tool joints, studies on the joints of ball screws, linear guides and bolts are carried out. The influence of preload on the axial stiffness of a ball screw is calculated based on Hertzian contact theory and the effect of pretightening moment on pressure of bolt joint is discussed, while the dynamic stiffness and the damping of a linear guide are identified by an optimum algorithm with the simulated and experimental results. A finite element model (FEM) of the whole machine tool structure considering the effects of different joints is created and verified against the test results, and then the influence of preloads on ball screws and linear guides is predicted. Results indicate that preloads on machine tool joints have significant effects on the dynamic stiffness of the spindle nose.