Objective: This study aimed to identify the clinical effectiveness of two different penetration depths of micro-osteoperforations (MOPs) on the rate of orthodontic tooth movement. Methods: Twenty-four patients requiring the removal of the upper first premolar teeth were selected and randomly divided into two groups. The control group participants did not undergo MOPs. Participants in the experimental group underwent three MOPs each at 4-mm (MOP-4) and 7-mm (MOP-7) depths, which were randomly and equally performed to either the left or right side distal to the canine. The retraction amount was measured on three-dimensional digital models on the 28th day of retraction. MOP-related pain was measured using a visual analog scale (VAS). Between-group statistical differences in the VAS scores were determined using an independent t-test and those in canine retraction were determined using analysis of variance and post-hoc Tukey test. Results: No significant difference was found between the MOP-4 (1.22 +/- 0.29 mm/month) and MOP-7 (1.29 +/- 0.31 mm/month) groups in terms of the canine retraction rate. Moreover, both the groups demonstrated a significantly higher canine movement than the control group (0.88 +/- 0.19 mm/month). MOPs did not significantly affect the mesialization of the posterior teeth (p > 0.05). Moreover, the pain scores in the MOP-4 and MOP-7 groups were similar and showed no statistically significant difference. Conclusions: Three MOPs with a depth of 4 mm can be performed as an effective method to increase the rate of tooth movement. However, three MOPs with depths of 4-7 mm does not additionally enhance tooth movement.