OBJECTIVES To examine the effects of mature adipocyte-derived dedifferentiated fat (DFAT) cell transplantation on urethral tissue regeneration and sphincter function. METHODS Sixteen female Sprague-Dawley rats underwent vaginal distension (VD) for 3 h. Subsequently, green fluorescence protein (GFP)-labeled DFAT cells (1×10(6) in 20 µL saline, DFAT group, n=8) or saline (20 µL, control group, n=8) were injected into paraurethral connective tissue. Two weeks following VD, leak point pressure (LPP) was measured and an immunohistochemical analysis of the urethra was performed to evaluate urethral sphincter regeneration. RESULTS The VD model was characterized by atrophy of the urethral sphincter and showed a decrease in LPP. DFAT cell transplantation resulted in a significant improvement of LPP (DFAT group: 37.3±6.4 vs control group: 21.7±5.7 mmHg, P<0.01). Immunohistochemistry revealed that the striated muscle thickness and smooth muscle α-actin-positive area were significantly (P<0.05) larger in the DFAT group than in the control group. DFAT cell transplantation enhanced macrophage accumulation followed by an increased number of cells in the proliferative state. Transplanted DFAT cells were observed in the damaged smooth muscle layer and showed positive staining for smooth muscle α-actin, suggesting conversion into the smooth muscle cell phenotype. CONCLUSIONS DFAT cell transplantation promotes sphincter muscle regeneration and improves LPP in the rat VD model.