Background Root canal shaping without any procedural error is of the utmost preference. Therefore, the purpose of this study was to use micro-computed tomography to evaluate and compare the root canal shaping efficacy of ProTaper rotary files and standard stainless steel K-files. Material/Methods Sixty extracted upper second premolars were selected and were divided into 2 groups of 30. Before preparation, all samples were scanned by micro-CT. Then, 30 teeth were prepared with stainless steel files and the remaining 30 with ProTaper rotary files. Canal transportation and centering ability before and after root canal shaping were assessed using micro-CT. The amount and direction of canal transportation and the centering ratio of each instrument were determined in the coronal, middle, and apical parts of the canal. The 2 groups were statistically compared using one-way ANOVA. Results ProTaper rotary files gave less transportation (p<0.001) and better centering ability (p<0.00001) compared with stainless steel files. Conclusions The manual technique for preparation of root canals with stainless steel files produces more canal transportation, whereas rotary files remain more centered in the canal.