The objective of the present in vitro work was to investigate the effectiveness and time required for the removal of calcium silicate-based sealer using two rotary retreatment systems. Sixty extracted, single-canal, lower premolars were used. After obturation using the single-cone technique with calcium silicate-based sealer, samples were divided into four groups according to the technique of desobturation: Group 1 (G1): D-Race; Group 2 (G2): D-Race followed by the use of XP–Endo Finisher R; Group 3 (G3): Protaper Universal Retreatment; and Group 4 (G4): Protaper Universal Retreatment followed by the use of XP–Endo Finisher R. Cone beam computed tomography (CBCT) images were used to calculate the remaining filling materials at the middle and apical thirds. Times required to perform each method were recorded. Scanning electron microscopy (SEM) and digital microscopy were used to evaluate the remaining filling materials. Data were statistically analyzed using the t-test and one way ANOVA on ranks tests. No statistically significant difference was found between G1 and G3 after CBCT observations (p > 0.05). Xp-Endo Finisher R significantly increased the ability to remove materials regardless of the initially used retreatment system (p < 0.05). Statistically significant longer time was found in G3 and G4 compared to G1 and G2, respectively (p < 0.05), to reach the full working length. No retreatment system was able to totally remove the calcium silicate-based sealer from the root canal at the middle and apical thirds (p > 0.05). Digital microscopy demonstrated that the residual materials were the remaining sealers on the canal walls. SEM showed the mineral depositions of calcium silicate materials onto the canal walls and into the dentinal tubules. However, that calcium silicate materials provide mineral deposition into the dentinal tubules might indicate that the traditional irrigants could not be sufficient to remove calcium silicate-based materials from the root canal, and other agents should be used to make retreatment considerably easier.