Objective： To evaluate the efficacy and security of two type of rotary nickel titanium system (Twisted File and ProTaper Universal) for root canal preparation based on micro-computed tomography(micro-CT).Methods: Twenty extracted molars (including 62 canals) were divided into two experimental groups and were respectively instrumented using Twisted File rotary nickel titanium system (TF) and ProTaper Universal rotary nickel titanium system (PU) to #25/0.08 following recommended protocol. Time for root canal instrumentation (accumulation of time for every single file) was recorded. The 0-3 mm root surface from apex was observed under an optical stereomicroscope at 25×magnification. The presence of crack line was noted. The root canals were scanned with micro-CT before and after root canal preparation. Threedimensional shape images of canals were reconstructed, calculated and evaluated. The amount of canal central transportation of the two groups was calculated and compared.Results:The shorter preparation time ［(0.53±0.14) min］ was observed in TF group, while the preparation time of PU group was (2.06±0.39) min (P<0.05). In mid-root level, TF group shaping resulted in less canal center transportation than PU group ［(0.070±0.056) mm vs. (0.097±0.084) mm, P<0.05］. No instrument separation was observed in both the groups. Cracks were not found in both the groups either based in micro-CT images or observation under an optical stereomicroscope at 25× magnification.  Conclusion:Compared with ProTaper Universal, Twisted File took less time in root canal preparation and exhibited better shaping ability, and less canal transportation.