Objective: The aim of the present study was to establish adequate protocols for scaffold generation and prepare for future in vivo sheep uterus bioengineering experiments. Uterus tissue engineering may dismantle limitations in current uterus transplantation protocols. A uterine biomaterial populated with patient-derived cells could potentially serve as a graft to circumvent complicated surgery of live donors, immunosuppressive medication and rejection episodes. Repeated uterine bioengineering studies on rodents have shown promising results using decellularized scaffolds to restore fertility in a partially impaired uterus and now mandate experiments on larger and more human-like animal models. Methods: Three decellularization protocols were developed using vascular perfusion through the uterine artery of whole sheep uteri obtained from slaughterhouse material. Decellularization solutions used were based on 0.5% sodium dodecyl sulphate (Protocol 1) or 2% sodium deoxycholate (Protocol 2) or with a sequential perfusion of 2% sodium deoxycholate and 1% Triton X-100 (Protocol 3). The scaffolds were examined by histology, extracellular matrix quantification, evaluation of mechanical properties and the ability to support fetal sheep stem cells after recellularization. Results: In all protocols, sheep uteri could successfully be decellularized while maintaining a high integrity of the extracellular components. Uteri perfused with sodium deoxycholate (Protocol 2) were the most favorable treatment in our study based on quantifications. However, all scaffolds supported stem cells for 2 weeks in vitro and showed no cytotoxicity signs. Cells continued to express markers for proliferation and maintained their undifferentiated phenotype. Conclusion: The present study shows three valuable decellularization protocols for future in vivo sheep uterus bioengineering experiments.