This study aimed to evaluate the procurement, efficacy, and applications of different acellular matrices (AMs) on rat esophagi. Sixteen rats were used. Following tissue harvesting from donor rats, AMs were obtained by detergent-enzymatic treatment (DET) from the esophagus, jejunum, and trachea. Fifteen rats were allocated into groups defined by implemented AM types: esophageal (EAM), intestinal (IAM), and tracheal (TAM). According to groups, 5 x 5 mm esophageal wall defect repair was performed with AMs. Samples were examined for hyperemia, edema, inflammatory cells, and neovascularization by scoring. The existence of muscle cells was investigated with immunohistochemical assessment. The procurement of AM in vitro and its implementation on the damaged esophageal wall was achieved successfully. There was a significant difference between groups regarding hyperemia, inflammatory cells, and neovascularization (p < 0.05). Hyperemia and cell infiltration were higher, and neovascularization was poorer in TAM than in others (p < 0.05). The neovascularization was significantly higher in the EAM group (p < 0.05). Increased edema was detected in the IAM group than others, albeit with no significant difference. Staining with Masson's trichrome and desmin antibodies, showing increased inflammatory response on the matrix site, was not detected to regenerate muscle cells. Our DET approaches provided that AMs with potential for regeneration have been obtained from different tissues. Grafting by TAM caused higher grades of inflammation with poor angiogenesis and existing chondrocytes. Decreased inflammatory response of EAM was thought to be due to the native tissue-derived matrix. These results suggest that promoting self-regeneration with native tissue-derived AM can be improved and more convenient in damaged tissue repair.