Spindle stiffness is one of the most critical indicators for evaluating and measuring the service performance of spindles. The traditional static stiffness indexes only involve static analysis and rarely focus on the study of spindle-carrying capacity under operating conditions. In this paper, the explicit solution approach is used to develop a mechanical model of the spindle’s axial operating stiffness. This model was then used to explore the influence of rotational speed on the softening and hardening features of the spindle axial operating stiffness, and experimental verification was carried out. According to studies, the speed of a fixed-position preload spindle can lead its operating stiffness to exhibit a “stiffness-hardening” feature. However, when the axial displacement of the spindle is small, the operating stiffness curve of the spindle displays a noticeable “fluctuation” phenomenon for low-speed spindles. Furthermore, the speed-induced preload has a significant impact on the test results when testing spindle axial operating stiffness.