TruNatomy (TN; Dentsply Sirona, Maillefer, Ballaigues, Switzerland) is a newly released system that was not tested in any previous studies. The objective of this work is to evaluate cyclic fatigue resistance of the new file and compare it with the Twisted Files (TF) and ProTaper Next (PTN). Forty-five files were distributed into 3 groups: PTN X2 (size 25 and taper 0.06), TF (size 25 and taper 0.06), and TN prime file (size 26 and taper 0.04). Each group included 15 files. Lengths of all files were 25 mm. Cyclic fatigue testing was done using artificial stainless-steel canals with 60-degree curvature and 5 mm radius. Continuous rotation movement at 300 rpm was used until the file fractures. Time for file separation was recorded in seconds. The number of cycles to failure (NCF) mean and standard deviation for each group was calculated. For statistical analysis of data, ANOVA and Tukey's multiple comparison test were used. Mean and standard deviation (SD) of NCF were 259 ± 37.2, 521.67 ± 63.07 and 846.67 ± 37.16 for PTN, TF, and TN respectively. TN on average had significantly the highest NCF compared with PTN ( < 0.05) and TF ( < 0.05). TruNatomy file showed superior cyclic fatigue resistance. With its potential to preserve tooth structure, this file offers a good cyclic fatigue advantage. However, future studies are required to evaluate other properties of this file and to examine its clinical performance.