PURPOSE: Pain, swelling, limitation of the mouth opening, development of intra-bony defects, and bone loss are common side effects of removing the impacted third molar. The purpose of this study was to measure the association of applying melatonin in the socket of an impacted mandibular third molar with osteogenic activity and the anti-inflammatory effects. METHODS: This prospective, randomized, blinded trial comprised of patients who required removal of the impacted mandibular third molar. The patients were divided into two groups (n = 19) as follows: melatonin group (3 mg of melatonin into 2 ml of 2% hydroxyethyl cellulose gel was packed into the socket) and placebo group (2 ml of 2% hydroxyethyl cellulose gel was placed in the socket). The primary outcome was bone density, measured using Hounsfield unit immediately after surgery and 6 months later. Secondary outcome variables included serum osteoprotegerin level (Ng/ml) that measured immediately, 4 weeks and 6 months postoperatively. Other clinical outcome measures were pain by visual analog scale, maximum mouth opening (MMO) (millimeter), and swelling (millimeter) that were evaluated immediately, 1, 3, and 7 days postoperatively. The data were analyzed by independent t-test of Wilcoxon's rank-sum, analysis of variance, and generalized estimating equation (P ≤ .05). RESULTS: Thirty-eight patients (25 female and 13 males) with a median age of 27 years were enrolled in the study. There was no statistical significance in bone density observed in both groups [melatonin group: 978.5(951.3-1015.8), control group: 965.8 (924.6-998.7), P = .1]. Alternatively, there were statistically significant improvements in osteoprotegerin levels (on week 4), MMO (on day1), and swelling (on day 3) in the melatonin group compared to those in the placebo group [1.9(1.4-2.4), 39.68 ± 1.35, and 14.36 ± 0.80 versus 1.5(1.2-1.4); 38.33 ± 1.20, and 14.88 ± 0.59; P = .02, .003, 0.031, respectively]. The pain values showed statistically significant improvement throughout the follow-up period in the melatonin group compared to the placebo group [5(3-8), 2(1-5), and 0(0-2) versus 7(6-8), 5(4-6), and 2(1-3); P < .001, respectively]. CONCLUSIONS: The results support the anti-inflammatory effect of melatonin in reducing the pain scale and swelling. Furthermore, it plays a role in the improvement of MMO. On the other hand, the osteogenic activity of melatonin could not be detected.