Introduction: The aim of this study was to determine the effect of thermal treatment on the flexural resistance of mechanically pre-fatigued rotary nickel-titanium (NiTi) endodontic instruments. Methods: New and mechanically pre-fatigued rotary NiTi instruments were subjected to thermal treatments at 350˚C, 400˚C and 450˚C for 30 minutes, and then were rotated in a fatigue-testing device until fracture. The time to fracture was recorded and multiplied by the number of rotations per minute to obtain the number of cycles to failure (NCF) for each group. One-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple-comparison post hoc test was used for the comparison between the different tested groups. Fractographic examination by scanning electron microscopy was also performed to study the morphological characteristics of the fractured instruments. Results: Statistical analysis showed that the thermal treatment at or below 400˚C of the new files did not improve the resistance to the flexural fatigue. Also, the flexural fatigue resistance of mechanically pre-fatigued files was significantly lower than that of new files. However, with thermal treatment significant improvement in the resistance to flexural fatigue was achieved, especially at 450˚C (P 0.05). Conclusion: Thermal treatment seems to be of significance in improving the cyclic fatigue resistance or life span of fatigued or used NiTi files. Keywords: flexural resistance, nickel titanium, thermal treatment