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X-ray computed micro-tomography scanning allows high-resolution 3-dimensional imaging of small objects. In this study, micro-CT scanning was used to compare the ability of manual and mechanical glide path to maintain the original root canal anatomy.Eight extracted upper first permanent molars were scanned at the TOMOLAB station at ELETTRA Synchrotron Light Laboratory in Trieste, Italy, with a microfocus cone-beam geometry system. A total of 2,400 projections on 360° have been acquired at 100 kV and 80 μA, with a focal spot size of 8 μm. Buccal root canals of each specimen (n = 16) were randomly assigned to PathFile (P) or stainless-steel K-file (K) to perform glide path at the full working length. Specimens were then microscanned at the apical level (A) and at the point of the maximum curvature level (C) for post-treatment analyses. Curvatures of root canals were classified as moderate (≤35°) or severe (≥40°). The ratio of diameter ratios (RDRs) and the ratio of cross-sectional areas (RAs) were assessed. For each level of analysis (A and C), 2 balanced 2-way factorial analyses of variance (P < .05) were performed to evaluate the significance of the instrument factor and of canal curvature factor as well as the interactions of the factors both with RDRs and RAs.Specimens in the K group had a mean curvature of 35.4° ± 11.5°; those in the P group had a curvature of 38° ± 9.9°. The instrument factor (P and K) was extremely significant (P < .001) for both the RDR and RA parameters, regardless of the point of analysis.Micro-CT scanning confirmed that NiTi rotary PathFile instruments preserve the original canal anatomy and cause less canal aberrations.