Nickel-titanium (NiTi) instruments are extensively used in endodontic treatment because of their outstanding mechanical properties. However, unexpected fracture of NiTi rotary instruments occurs during endodontic procedures. Therefore, a reliable method to detect the structural status of a used NiTi instrument is needed. The aim of this study is to use natural frequency for monitoring structural changes of a NiTi instrument during and after the instrumentation process. In this study, laboratory modal testing experiments were performed on cyclic fatigue-loaded NiTi rotary instruments with a natural frequency detecting device. In addition, three-dimensional finite element (FE) models were established for assessing the structural changes that take place in repeatedly loaded NiTi instruments. Repeated rotational loading resulted in a significant decrease (p < 0.05) in natural frequency (with a decreasing ratio of 5.6%) when the tested instruments reached 77-85% of their total life limit. In FE analysis, a strong correlation between natural frequency and change in elastic modulus of the NiTi instrument was found. These findings indicated that natural frequency may represent an effective parameter for evaluating the micro-structural status of NiTi rotary instruments subjected to fatigue loadings.