Abastract: Background and Aim: Calcium hydroxide (CH) is one of the most common intracanal medicaments. The residual calcium hydroxide along the root canal wall increases or decreases the sealing quality of the root canal filling. This study aimed to compare the leakage of different sealers in presence of calcium hydroxide. Materials and Methods: In this in-vitro study, the 90 three-rooted and canal human mandibular molar teeth with mature apex selected with a curvature of less than 10 degrees were cleaned and shaped and divided into two groups (n=45). The first group was not medicated and the second group was medicated with calcium hydroxide. After 2 weeks, calcium hydroxide was removed from the root canal. Then the root canals were sealed using 3 different types of sealer (MTA Fillapex, AH26 and Endoseal MTA) with standard gutta percha and lateral condensation technique. The samples were immersed in India ink. After 4 weeks, the samples were demineralized and dehydrated. Apical leakage was measured by a microscope with a magnification of 0.1 mm. Data were analyzed using SPSS software and two-way ANOVA and Tukey HSD test Results: The lowest levels of leakage, both in the presence and absence of calcium hydroxide, were observed in the AH26, Endoseal MTA, and MTA Fillapex sealers, respectively. The leakage level in each sealer was lower in the absence of calcium hydroxide compared to its presence. Based on the two-way ANOVA test, the type of sealer (P<0.001), the use or non-use of calcium hydroxide (P<0.001), and the interaction between the type of sealer and the use or non-use of calcium hydroxide (P<0.001) had a significant impact on leakage. The mean leakage of the AH26 sealer was significantly lower than that of the Endoseal MTA and MTA Fillapex sealers (P<0.001). The mean leakage of the Endoseal MTA sealer was significantly lower than that of the MTA Fillapex sealer (P<0.001). Conclusion: All sealers had apical leakage and filling with them with and without the calcium hydroxide did not completely prevent dye leakage. The leakage rate of AH26 was lower than the other two sealers and the leakage rate of MTA Endosael was lower than MTA Fillapex. In the absence of calcium hydroxide, the amount of leakage is less.