Preservation of the original anatomical shape following instrumentation of root canals is essential for endodontic success. Procedural errors created during glide path enlargement might be exacerbated or initiated during subsequent shaping. The aims of this study were to: (1) compare canal centering ability and transportation of pre-curved Senseus K-FlexoFiles (stainless steel), ProGlider file (M-Wire) and One G file(NiTi alloy) after glide path enlargement in curved root canals micro-computed tomography (micro-CT) scanning; (2) compare canal centering ability and transportation of OneShape (NiTi alloy), ProTaper NEXT (M-Wire alloy) and WaveOne Gold (Gold wire) instrumentation techniques in the same canals; (3) compare the change in root canal volume between uninstrumented canals, canals after glide path preparation, and canals after root canal preparation. One hundred and thirty-five curved mesio-buccal root canals of human maxillary molars were randomly divided into three groups. These groups were (1) glide path enlarged using pre-curved size 10, 15 and 20 stainless steel Senseus K-FlexoFiles (n=45); (2) manual glide path enlargement with a size 10 K-File followed by One G (n=45); and (3) manual glide path enlargement with a size 10 K-File followed by the ProGlider (n=45). Micro-CT was used to scan teeth before and after glide path preparation. Each glide path specimen group was randomly assigned to three equal groups (n=15) resulting in nine glide path/shaping groups of fifteen canals each: Group 1 (K-FlexoFile + OneShape)(K/OS); Group 2 (K-FlexoFile + ProTaper NEXT)(K/PTN); Group 3 (K-FlexoFile + WaveOne Gold)(K/WOG); Group 4 (One G + OneShape)(OG/OS); Group 5 (One G + ProTaper NEXT)(OG/PTN); Group 6 (One G + WaveOne Gold)(OG/WOG); Group 7 (ProGlider + OneShape)(PG/OS); Group 8 (ProGlider + ProTaper NEXT)(PG/PTN); and Group 9 (ProGlider + WaveOne Gold)(PG/WOG). After canal preparation with the shaping instruments, all the specimens were scanned again by means of micro-CT. The three-dimensional images obtained before instrumentation, after glide path preparation, and again after final canal preparation were reconstructed and interpreted. Centering ratio values, canal transportation and change in root canal volume were recorded and compared between the three glide path- and nine root canal preparation groups. Canal transportation and centering ability were evaluated over the apical, midroot, and coronal levels (2 mm, 5 mm and 9 mm from the root apex). The results were statistically analysed using a one-way ANOVA for parametric and Kruskal-Wallis H test for non-parametric comparisons. Statistical significance was set at p< 0.05. One G and ProGlider displayed statistically significantly better mean centering ratios than stainless steel K-FlexoFiles at each level examined and for the combined results of the three levels (p<0.05). Apical canal transportation ratio values after glide path enlargement were significantly higher for the K-File group compared to One G and ProGlider (p<0.05). At the midroot and coronal levels and for the combined results of the three levels, the canal transportation results were statistically similar for all glide path groups (p>0.05). The volume of dentine removed by the three glide path groups was statistically significantly similar for K-FlexoFiles, One G and ProGlider (p<0.05). No statistically significant difference was found in the mean centering ratios at the apical and midroot levels of the various glide path groups in combination with the shaping instruments (p>0.05). However, at the coronal level, centering ratio results following glide path preparation with K-FlexoFiles appeared to affect shaping outcomes for both PTN and OS groups. One Shape performed poorly following all glide path techniques with OG/OS and significantly displayed the worst centering ratio at this level. The results for the combined centering ratio values of the various glide path/shaping groups displayed no statistically significant differences between the different combination groups (p>0.05). Apical canal transportation after shaping was significantly highest for K/OS followed by K/PTN. At the midroot level canal transportation was significantly higher for K/PTN than K/OS and OG/OS, which were statistically similar to each other. Coronal canal transportation after canal shaping was significantly highest for K/PTN followed by K/OS. The most favourable mean combined transportation ratio values of the various glide path/shaping groups were observed in OG/WOG and in PG/WOG groups and the least favourable for the K/OS and the K/PTN groups. The three groups shaped with ProTaper NEXT exhibited the highest volume of dentine removed with the highest displayed by the PG/PTN group. Statistically, the lowest mean volume of removed dentine was by the PG/WOG group. In general, results were more favourable after canal preparation with the WaveOne Gold Primary file following any of the three glide path preparation techniques.