Engineering of urinary bladder has been the focus of numerous studies in recent decade. Novel biomaterials, innovative fabrication methods and various modification processes of scaffolds are the critical issues to find supportive matrices. Supportive characteristics of electrospun PCL/PLLA nano-scaffold for bladder augmentation in canine model and the role of bladder cells in regeneration process were appraised. Electrospun PCL/PLLA was fabricated by co-electrospinning of PCL and PLLA. Bladder cells were isolated and transduced with lentiviral particles encoding eGFP and JRed proteins. Electrospun PCL/PLLA was seeded with different bladder cells individually or in co-culture condition. Cell-free and cell-seeded electrospun PCL/PLLA scaffolds (10 cm2) were surgically implanted in bladders of eight female dogs for three months. To evaluate bladder regeneration, the dogs were sacrificed and their bladders were examined macroscopically and microscopically for presence of tracking proteins, expression of cell-specific markers and histological attributes of regenerated tissues. All animals survived the experiment with no complication. In smooth muscle transplanted group complete regeneration and covering of scaffold were observed. Other groups revealed partial regeneration. A well-developed layer of urothelium was formed in all groups in regenerated parts. Smooth muscle transplanted group showed the most developed muscle layer. Regenerated tissue demonstrated typical expression of cell-specific markers. No expression of eGFP and JRed was observed. Electrospun PCL/PLLA scaffold with proper handling, suture retention, nano-sized surface features, maintenance of normal phenotype of cells and minimal adverse effects in body can be a supportive substrate for bladder wall regeneration when seeded with bladder smooth muscle cells.