Cadaveric organ transplantation represents the definitive treatment option for end-stage disease but is restricted by the shortage of clinically-viable donor organs. This limitation has, in part, driven current research efforts for in vitro generation of transplantable tissue surrogates. Recent advances in organ reconstruction have been facilitated by the re-purposing of decellularized whole organs to serve as three-dimensional bio-scaffolds. Notably, studies in rodents indicate that such scaffolds retain native extracellular matrix components that provide appropriate biochemical, mechanical and physical stimuli for successful tissue/organ reconstruction. As such, they support the migration, adhesion and differentiation of reseeded primary and/or pluripotent cell populations, which mature and achieve functionality through short-term conditioning within specialized tissue bioreactors. Whilst these findings are encouraging, significant challenges remain to up-scale the present technology to accommodate human-sized organs and thereby further the translation of this approach towards clinical use. Of note, the diverse structural and cellular composition of large mammalian organ systems mean that a “one-size fits all” approach cannot be adopted either to the methods used for their decellularization or the cells required for subsequent re-population, to create fully functional entities. The present review seeks to highlight the clinical potential of decellularized organ bio-scaffolds as a route to further advance the field of tissue- and organ-regeneration, and to discuss the challenges which are yet to be addressed if such a technology is ever to become a credible rival to conventional organ allo-transplantation.