BACKGROUND Rotary endodontic instruments are increasingly used in root canal treatment and have replaced stainless steel manual files. Cyclic fatigue is the cyclic loading of stress to produce deformation or fracture. This study aimed to evaluate and compare the effects of autoclave sterilization on cyclic fatigue in 5 types of rotary endodontic instruments. MATERIAL AND METHODS ProTaper Universal, BioRace, ProTaper Next, Twisted File, and HyFlex CM instruments were included in this study. Each type included 96 instruments, divided into 4 groups according to the number of sterilization cycles (0, 1, 3, 5). After sterilization, each group of instruments was divided into 2 subgroups and tested for cyclic fatigue in 2 simulated canals (45 degrees both and 2 radii, 2 mm and 5 mm). The number of cycles to failure (NCF) was calculated, and statistical analyses were carried out using the t test, Mann-Whitney U test, and ANOVA, followed by the Tukey post hoc test (p<0.05). Fracture surfaces were analyzed using scanning electron microscopy (SEM). RESULTS Within the group of non-sterilized instruments, ProTaper Universal showed significantly lower resistance to cyclic fatigue compared to the other types of instruments (p<0.001). After repeated sterilization, a significantly higher mean of NCF was observed for BioRace (p<0.001), ProTaper Next (p<0.001), Twisted File (p<0.001), and HyFlex CM (p<0.001) compared to ProTaper Universal. The resistance of HyFlex CM was significantly higher compared to the other types of instruments (p<0.001). CONCLUSIONS The findings from this study showed that autoclave sterilization of newer rotary endodontic instruments could increase resistance to cyclic fatigue.