Introduction: Root canal retreatment procedure may reduce the mechanical resistance of nickel-titanium instruments and increase the frequency of instrument fracture. The aim of the present study was to evaluate the effect of multiuse of Reciproc Blue R25 instruments on cyclic fatigue resistance during retreatment procedure in mandibular molar teeth. Methods: Forty-eight Reciproc Blue R25 files were included in the study. In control group, 12 new Reciproc Blue R25 instruments were subjected to cyclic fatigue test by using a stainless steel artificial canal with a 90 degrees angle and 3-mm radius of curvatures. In experimental groups, Reciproc Blue R25 files (n = 12) were used to remove the root canal filling (RCF) material of 1, 2, and 3 molar teeth, respectively. Then cyclic fatigue tests of these 36 instruments were performed. The number of cycles to fracture (NCF) and the length of the fractured fragment were calculated for each instrument. Working time for retreatment procedures was determined in seconds with a digital chronometer. The data were analyzed by using one-way analysis of variance followed by post hoc Tukey test and mixed-effect analysis (P = .05). Results: No significant difference was found between NCF values of new and first time used files (P > .05), but NCF values of new and first time used files were significantly higher than those of second and third time used files (P < .05). The second and third time used files revealed no difference (P > .05). As the number of uses increased, the average time for retreatment procedure statistically extended (P < .05). Conclusions: Reciproc Blue R25 instrument can be used to remove RCF material of 2 molar teeth. However, because of its decreased NCF values, multiple use over 2 molars may not be suggested.