Background. The main goal of our study was to assess the volume of dentin removed and transportation in root canals using Protaper Universal (PTU), Protaper Gold (PTG) and One-Curve (OC). Ni-Ti rotary instruments in extracted human teeth using by micro-CT. Methods: Thirty human upper 1st premolar teeth with two separate root canals and sturdy, mature root tips were used in the present study. Specimens were decoronated and root length was standardized for micro CT scanning before root canal preparation done. The teeth were randomly separated into three categories (n = 10) according to the rotary NiTi system used for canal instrumentation, i. e., PTU (Dentsply, Maillefer), PTG (Dentsply, Maillefer), and OC (Micro-Mega SA). After root canal preparation, samples were scanned again on micro-CT by the same scanning parameters. Surface area, canal volume, structure model index (SMI), percentage of uninstrumented area and transportation parameters were obtained for each sample before and after micro-CT analyse. Results: No significant differences between the PTG and PTU in terms of the total volume of removed dentin, surface area and percentage of uninstrumented areas were found. However, regarding to parameters above, OC showed a lower efficacy than PTG and PTU in coronal section. Regarding canal transportation, PTG and OC showed lower mean transportation values at all levels. Conclusion: This paper demonstrated the root canal shaping abilities of the PTU, PTG, and OC NiTi file systems. The PTG and OC systems were associated less canal transportation and a better ability to preserve dentinal walls than PTU. There was no significance different between all rotary file systems for SMI values however, PTU and PTG showed greater canal volume and surface area change than OC file systems in coronal section.