Objective: This study compared the postoperative pain after the use of manual and rotary instrumentation of the same instrument design, namely ProTaper Universal system (Dentsply Maillefer, Tulsa Dental, TN, USA) in mandibular posterior teeth after single root canal treatment, in a randomized clinical trial.Participants and Methods: Forty-six participants with vital or non-vital, mandibular posterior teeth without periapical pathosis, were endodontically treated in single visit. The patients were equally allocated at random into two groups. The instrumentation was done according to manufacturer instructions using either manual ProTaper files for group 1 or rotary ProTaper files for group 2. Irrigation was done using 2.6% NaOCl and 17% EDTA, canals were obturated using gutta-percha and resin sealer by active lateral compaction technique. Pain scoring was done using Numerical Rating Scale (NRS) preoperatively then after 6 hours, 12 hours, 24 hours, 48 hours, 72 hours and 7 days postoperatively. Pain levels were assigned to one of four categories, None, Mild, Moderate or Severe. Statistical analysis was done by chi square (c2) test and Fisher’s exact test when appropriate, P-values < 0.05 were considered significant. Results: Root canal preparation using manual ProTaper files was associated with statistically higher moderate and severe postoperative pain categories in comparison with rotary ProTaper files in the first 24 hours, P<0.05, which led to patients taking more analgesic tablets, P<0.05. Both groups had a significant decrease of postoperative pain in relation to preoperative value, P<0.001. While postoperative pain in rotary group started to decrease after 6 hours, in manual group it started to decrease after 12 hours. Conclusion: Rotary instrumentation results in a lower incidence and a shorter duration of postoperative pain than manual instrumentation.