Introduction: The aim of this study was to compare the transportation and centering abilities of the ProGlider (PG; Dentsply-Sirona, Ballaigues, Switzerland), WaveOne Gold Glider (WOGG; Dentsply-Sirona), and R-Pilot (RP; VDW, Munich, Germany) file systems and the changes in the structure model index (SMI), surface area, and root canal volume (after creating the glide path in the mandibular first molar teeth's mesial canals with moderate curvature) using the micro-computed tomographic (micro-CT) method. Methods: In the present study, 24 mandibular first molar teeth with 2 separate mesial canals and moderate (10 degrees-20 degrees) curvature were used. After scanning with micro-CT imaging before the procedure, the teeth were divided into 3 groups, PG, WOGG, and RP (n = 10/each group), followed by the glide path procedure. After the root canal preparation, changes in the root canals regarding the volume, surface area, SMI, transportation made by the files, and the change of their centering ability in the apical, middle, and coronal third were analyzed by micro-CT imaging. The data were statistically evaluated using 1-way analysis of variance and the Kruskal-Wallis test with a significance level set at 5% (P < .05). Results: The WOGG and RP file systems showed better centering abilities at all thirds compared with the PG file system (P < .05). No significant difference was found in the apical third between the groups in terms of canal transportation degree (P > .05). However, in the WOGG and RP groups, less transportation was observed in the middle and coronal thirds compared with the PG group (P < .05). When the SMI changed, after the root canal preparation was compared, no significant differences were found between the groups (P > .05). Nevertheless, the changes in volume and surface area of the root canal in the WOGG and RP groups were significantly lower than in the PG group (P < .05). Conclusions: The WOGG and RP file systems caused similar levels of change in the root canal systems based on all parameters. Moreover, it was determined that the PG file system caused a significantly higher volume and surface area increase than the WOGG and RP file systems.