Objective(s): The aim of this study was to evaluate the effect of manual, rotatory, and reciprocating endodontic instrumentation for uniradicular teeth on fatigue, load to failure, and dentin crack formation. Materials and Methods: Sixty-two human uniradicular teeth were selected. Root samples were standsdized at 14 mm (t0). Teeth were divided into three groups according to endodontic instrumentation: Manual (M), Rotatory (RT), or Reciprocating (RC) and filled with passive technique (t1). All teeth received the cementation of a glass fiber post and were restored with composite resin core (t2) and metallic crown. Samples were subjected to mechanical cycling (177 N, 2x106 cycles, 4 Hz) (t3), followed by a load to failure test. Failure analysis was performed and dentin crack analysis was conducted in two samples each time t0, t1, t2, and t3. Results: Load to failure was not different between groups (p=0.716). However, the M group presented more irreparable failures. M and RC groups presented a high percentage of root sections with defects, and t3 was the evaluation time which presented more dentin crack formation. Conclusion(s): Endodontic instrumentation system did not affect load to failure of uniradicular teeth. Still, it affected the failure mode, with manual and reciprocating instrumentations presenting more irreparable failures and high root dentin crack formation.