Acuseal arteriovenous graft is an early cannulation graft having a tri-layered structure with an elastomeric middle layer. However, delamination of Acuseal grafts has been reported recently. This article describes two cases with different characteristics of Acuseal delamination. In case 1, the delamination occurred 1 month after a percutaneous transluminal angioplasty (PTA); therefore, the PTA was suspected to be a trigger. The delamination was located between the outer expanded polytetrafluoroethylene (ePTFE) layer and the elastomeric middle layer. On the other hand, in case 2, the delamination was located between the luminal ePTFE layer and the elastomeric middle layer. A surveillance ultrasound examination detected the delamination unexpectedly in the uneventful course; however, the delaminating location corresponded to the cannulation puncture site and the intraoperative findings suggested the involvement of mis-needling as a possible cause. Interestingly, for the purpose of continued use on hemodialysis, specific treatments were required against the delamination itself in both cases. As we identified Acuseal delamination in 5.6% (2/36) of cases, concerns arise that numerous cases of Acuseal delamination may have been overlooked in general. Understanding and recognizing this phenomenon are important for adequate use of Acuseal graft.