Introduction: The purpose of this study was to compare the cyclic fatigue resistance of used and new ProGlider (Dentsply Maillefer, Ballaigues, Switzerland) and PathFile (Dentsply Maillefer) rotary glide path files. Methods: Forty ProGlider and 40 PathFile #2 instruments were used in the present study. In group 1, 20 PathFile #2 files and, in group 2, 20 ProGlider files were used to prepare a total of 40 J-shaped transparent acrylic blocks. In group 3 (control group), 20 new Path File files and, in group 4 (control group), 20 new ProGlider files were included. Cyclic fatigue testing of instruments was performed in a stainless steel artificial canal with a 5-mm radius of curvature and a 60 angle of curvature. All 80 instruments were rotated until fracture, and the number of cycles to failure (NCF) was recorded. Data were analyzed using 1-way analysis of variance to determine any statistical difference; the significance was determined at the 95% confidence level. Results: The used glide path files' NCF was lower than the new ones; however, this difference was not statistically significant (P > .05). The cyclic fatigue resistance of the used and new PathFile #2 files was statistically significantly higher than that of the ProGlider files (P < .05). Conclusions: Within the limitations of the present study, the used glide path files' NCF, was lower than the new ones; however, this difference was not statistically significant.