Ship’s propulsion shafting is one of the main sources of ship vibration and noise. The shafting, whirling vibrations, and alignment are important factors that affect the comfort, stability, and reliability during a ship’s navigation. However, the mechanism of the interacting of the both factors is not fully revealed. In this paper, the effect of shafting alignment on whirling vibration and the bearing temperature response is studied by experiment. The test scheme is designed reasonably according to the theoretical analysis. The results show that the horizontal component of the shafting whirling vibration can be effectively reduced by adjusting the shafting alignment state while the vertical component is not. The shafting axis balancing position (SABP) slightly moves upward in high speed, which should be considered in the dynamic alignment design of the shafting, especially for the high-speed shafting. Little ABSB (the angle between the shafting centre line and the No. 1 bearing centre line) is beneficial to the stable operation of shafting, while appropriately increasing the ABSB and bearing load is beneficial to reducing the shafting whirling vibration. By balancing the relationship between bearing load and ABSB, the performance of whirling vibration and bearing temperature response can be optimized.