This study assessed the outcome of shaping the mesial roots of mandibular first molars containing band-shaped isthmuses with an isthmus floor located at the apical third with Reciproc Blue and Rotate instruments in terms of canal transportation, canal aberrations and unprepared apical canals. Pair-matched mesial roots of mandibular first molars according to their isthmus location (distance from apical foramina) and size were constructed based on quantitative measurements (length, major and minor diameter at isthmus roof and floor) on preoperative micro-CT scanning (n = 10). The pair-matched specimens were prepared either using Reciproc Blue R25 (25.08v) or Rotate (15.04, 20.05, 25.06) instruments and then subjected to a second micro-CT scanning. Micro-CT scans were analysed to evaluate the canal transportation at the apical foramen and isthmus floor levels and the frequency of procedural errors (separated instruments, artificial canal preparation, cross shaping and unprepared apical canals). Canal transportation values were compared using Mann-Whitney U, and the distribution of procedural errors was compared with chi-square tests with a 5% significance threshold. Greater transportation values were detected in the apical foramen level compared to the isthmus floor in both groups. No significant difference was detected between Rotate and Reciproc Blue groups in terms of absolute transportation values and the incidence of procedural errors (p > .05). Preparation of mesial root canals containing a band-shaped isthmus at the apical portion with Reciproc Blue and Rotate instruments caused transportation of the original canal position at the isthmus floor and the apical foramen levels and resulted in procedural errors. This ex vivo micro-CT study using controlled pair-matched specimens showed that presence of a band-shaped isthmus complicates canal preparation procedure irrespective of the preparation systems.