Background & Objectives: The present investigation was aimed to assess the effect of pre-topical fluoride therapy on the microleakage of fissure sealants in permanent teeth. Materials and Methods: In this interventional in-vitro study, 45 molar teeth without decay were assessed. Samples were randomly divided into 3 groups with 15 cases. In groups, one and two, acidulated phosphate fluoride (AFP) topical gel was applied for four minutes. After 30 minutes, sealant therapy was performed on the occlusal surface of molar teeth. In group three (control), samples were treated with sealant therapy only. The depth of dye penetration was assessed using a calibrated stereomicroscope at 40x magnification. Data analysis was performed using the non-parametric Kruskal-Wallis test, and a p-value < 0.05 was considered significant. Results: In the present study, no statistically significant difference was observed between the study groups regarding the microleakage of fissure sealants and use of AFP topical gel. Conclusion: According to the results of this investigation, use of APF gel (1.23%, pH=3.5) before sealant therapy had no effect on microleakage of fissure teeth sealed with Pulpdent resin sealant (P=0.052). Therefore, fluoride treatment could be performed before sealant therapy.