Statement of the Problem: File fracture can interfere with cleaning and shaping of the canal and compromise periradicular healing. Autoclave sterilization may prone the files to fracture.Purpose: The purpose of the present study was to determine the effect of autoclave sterilization on the cyclic fatigue resistance of Hero642 rotary instrument in two curvatures of 45 and 60 degrees.Materials and Method: For this experimental in-vitro study, 90 Nickel-Titanium HERO 642 rotary files #30 with 0.06 taper were selected. They were divided into two groups (curvature of 45 and 60 degree) of 45 files. Each group was then subdivided into 3 subgroups; group I: no sterilization, group II: 5 cycles of sterilization and group III: 10 cycles of sterilization. Files were used in artificial canals until fracture. The cyclic fatigue was measured as the number of cycles before fracture. The data was statically analyzed by Student’s t-test and two-way analysis of variance.Results: There was a significant difference in cyclic fatigue of two curvature of 45 and 60 degrees (p= 0.001). However, sterilization process has no significant effect on cyclic fatigue of HERO files (p= 0.557).Conclusion: Sterilization had no effect on the cyclic fatigue of HERO 642 files when used in curvature of 45 or 60 degrees.