Modern machine tools are required to operate at wide speed ranges for higher productivity and quality. Because spindle bearings generate a large amount of heat at high speeds, the compensation for thermal effects in machine tools becomes exceedingly important. In this paper, a preload model is developed to describe how external cooling (or heating) affects the bearing preload. Based on the model, an active thermal preload regulation scheme is proposed and its feasibility is validated experimentally. The proposed preload regulation scheme is achieved by circulating a cooling (or heating) flow around the spindle housing to manipulate the housing and the outer ring temperatures. The results show that thermal preload is highly sensitive to the spindle outer ring and housing temperatures, and with proper preload level regulated by the cooling flow, the bearing runs steadier with less thermal preload fluctuations and is less disturbed by external loading. The results are significant for modern high performance spindle designs where different preload levels are required to cope with different machining conditions and where cooling is adopted to remove the large amount of heat produced at high speeds.