Purpose: The purpose of this study was to evaluate the effect of occlusal reduction and some other factors and conditions on post-operative pain levels during endodontic treatment.Methods & Materials: In this experimental study, one hundred & twenty one patients requiring endodontic treatment in posterior teeth were selected. Specific clinical findings including gender, age, pulp vitality, sensitivity to percussion, preoperative pain, presence of periradicular radiolucency, swelling, and bruxism were recorded. Teeth with restoration to be maintained, greater than class I mobility, pocket depth >5mm, endodontic retreatment, endodontic failures such as root perforation and over instrumentation, and patients who had less than 3 occluding teeth in each arch and the ones taking pain altering medications were excluded from the study. Then canal instrumentation was done. Teeth were randomly assigned to 1 of 3 groups: (i) total occlusal reduction, (ii) simulated occlusal reduction, (iii) Control (occlusion untouched). A questionnaire was used to assess pain responses over a 24h postoperative period. The results were analysed using a χ2 test (p<0.05).Results: A significant differences were seen among 3 occlusal treatment groups and the patients' pain responses (P<0.05). Significant lower persentage of patients reported moderate and severe pain (17%) in total occlusal reduction group in comparison with simulated occlusal reduction (41%) and occlusion untouched (45%) groups. Also a significant relationship was found between the following preoperative conditions and post-instrumentation pain: Gender, tooth type, pulp vitality, preoperative pain, periradicular radiolucency.Conclusion: Females requiring endodontic treatment in molar with necrotic pulps, presence of preoperative pain and the absence of periradicular radiolucency experience more pain after instrumentation than other patients. Occlusal reduction is recommended in patients having all or some of the mentioned preoperative conditions to decrease patients' pain and discomfort.