To evaluate the ability of ProGlider instruments, PathFiles and K-files to maintain canal anatomy during glide path preparation using X-ray computed micro-tomography (micro-CT). Forty-five extracted maxillary first permanent molars were selected. Mesio-buccal canals were randomly assigned (n=15) to manual K-file, PathFile or ProGlider groups for glide path preparation. Irrigation was achieved with 5% NaOCl and 10% EDTA. After glide path preparation, each canal was shaped with ProTaper Next X1 and X2 to working length. Specimens were scanned (isotropic voxel size 9.1μm) for matching volumes and surface areas and post-treatment analyses. Canal volume, surface area, centroid shift, canal geometry variation through ratio of diameter ratios and ratio of cross-sectional areas were assessed in the apical and coronal levels and at the point of maximum canal curvature. One-way factorial anovas were used to evaluate the significance of instrument in the various canal regions. Post-glide path analysis revealed that instrument factor was significant at the apical level for both the ratio of diameter ratios and the ratio of cross-sectional areas (P<0.001), with an improved maintenance of root canal geometry by ProGlider and PathFile. At the coronal level and point of maximum canal curvature, ProGlider demonstrated a tendency to pre-flare the root canal compared with K-file and PathFile. PathFile and ProGlider demonstrated a significantly lower centroid shift compared with K-file at the apical level (P=0.023). Post-shaping analysis demonstrated a more centred preparation of ProGlider, compared with PathFile and K-files, with no significant differences for other parameters. Use of ProGlider instruments led to less canal transportation than PathFiles and K-files.