Abstract: Aiming at the problem of poor consistency of residual pre-tightening force of bolt group in CNC machine tools assembly, an experimental bench was set up to study the effect of tightening sequence and step tightening strategies on residual pre-tightening of bolt group. First, based on the common four-bolt connection structure in machine tools, the experiment part of four-bolt connection structure is designed and fabricated. Secondly, an experiment platform was built to analyze the influence of tightening sequence and step tightening strategy on the residual pre-tightening force of the four-bolt connection structure. Finally, the two step tightening strategies are compared and analyzed. The results show that: among the three tightening sequences, the residual pre-tightening force consistency of the four-bolt connection structure is the best under the clockwise tightening condition. With the increase of tightening steps, the consistency of residual pre-tightening force of bolt group becomes better, but with the continues to increase of tightening steps, the consistency of residual pre-tightening force does not improve significantly. The consistency of the residual pre-tightening force of the bolt group becomes better with the increase of the first step tightening proportion, while the consistency of the residual pre-tightening force does not improve significantly with the continuous to increase of the first step tightening proportion. This study can provide guidance for improving machine tools assembly quality and bolt pre-tightening effect.