The aim of this experimental study focused on the relationship between pull-out strength (POS) and interfragmentary compressive force (IFCF) of AO cancellous lag screw during tightening procedure. The 6.5 mm AO cancellous lag screw and synthetic cancellous bone were used for this research. The test contains rotation tests and the subsequent pull-out tests, to record the IFCF and POS under different tightening angle groups. The results of this study demonstrated the specific relationship between IFCF and POS and showed that they didn’t reach the peak at the very same time. This study revealed the change of mechanical environment surrounding AO lag screw during tightening procedure and found the effective method to determine the optimum terminating time of AO lag screw inserting.