Biocompatible, biodegradable, and biomimetic scaffolds in combination with stem cells are of great importance for tissue engineering, especially the repairing and regeneration of defected organs. As a case in point, esophageal diseases have become serious clinical problems because of the poor self-repairing ability of the organ. It is crucial to prepare artificial replacements with biological function for serious lesions of the esophagus. However, the pH value, mechanical strength, thickness, and other physical conditions are very different in different organs or different parts of the same organ, which pose high difficulty for successful tissue engineering. In this work, bone marrow mesenchymal stem cells (bMSCs) were isolated from rabbits and transfected with green fluorescent protein (GFP) to follow their capabilities of growth, stemness, and differentiation in ex vivo culture. These bMSCs were seeded on biocompatible, biodegradable, and biomimetic scaffolds to detect the tissue regenerative capability of the esophagus with multilayer hierarchical structure. According to the esophageal bilayer muscle architecture, we designed discontinuous and continuous microchannel patterned scaffolds with medical level polyurethane (PU) as the matrix to guide the inner-circular and outer-longitudinal muscle growth. The gap on the discontinuous walls not only helped cells to communicate with each other but also assisted cells to infiltrate through the gap and grew into the inner circular muscle. The graft of silk fibroin on the scaffold surface using the aminolysis and glutaraldehyde cross-linking method enhanced the substrate's hydrophilicity and biocompatibility. Mucosa-submucosa tissue of rabbit's esophagus was decellularized to obtain the extracellular matrix (ECM) and implanted in situ after recellularizing with bMSCs to repair the partially defected rabbits' esophagus. On the basis of both in vitro and in vivo results, we concluded that esophagus regeneration was promoted by the differentiation of bMSCs on the biocompatible, biodegradable, and biomimetic scaffolds, starting from tissue "niches", to repair the largely defected esophagus, which paves the way for tissue engineering and defected organ treatments.