The aim of this ex vivo study was to evaluate the effect of using a patency file on apical transportation and curve straightening during canal instrumentation with the ProTaper rotary system. Seventy permanent mandibular first molars with mesiobuccal canals, measuring 18-23 mm in length and with a 25-40° curvature (according to the Schneider method), were selected. The working lengths were determined and the teeth were mounted and divided into two experimental groups: (A) prepared by the ProTaper system without using a patency file (n = 35) and (B) prepared by the ProTaper system using a patency file (n = 35). Radiographs taken before and after the preparation were imported into Photoshop software and the apical transportation, and curve straightening were measured. Data were analyzed using independent t-test. Partial correlation analysis was performed to evaluate the relationship between the initial curvature, transportation, and curve straightening (α = 0.05). Using a patency file during canal preparation significantly decreased both apical transportation and curve straightening (P < 0.001). There were significant relationships between the angle of curvature, transportation and curve straightening in pairs (P < 0.001). Apical patency is recommended during root canal preparation with the ProTaper rotary system.