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 decellularised scaffolds to restore fertility in a partially impaired uterus, and now mandate experiments on larger and more human-like animal models. The aim of the presented studies was therefore to establish adequate protocols for scaffold generation, and prepare for future in-vivo sheep uterus bioengineering experiments. Three decellularisation protocols were developed using vascular perfusion through the uterine artery of whole sheep uteri obtained from slaughterhouse material. Decellularisation solutions used were based on 0.5% sodium dodecyl sulfate (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 recellularisation. We showed that a sheep uterus can successfully be decellularised while maintaining a high integrity of the extracellular components. Uteri perfused with sodium deoxycholate (protocol 2) was the most favourable treatment in our study based on quantifications. However, all scaffolds supported stem cells for two weeks in vitro and showed no cytotoxicity signs. Cells continued to express markers for proliferation and maintained their undifferentiated phenotype. Hence, this study reports three valuable decellularisation protocols for future in-vivo sheep uterus bioengineering experiments.