Background: Tissue engineering integrating cell-based approaches is promising for urinary tract injury treatments. Purpose: Exploring the Impact of Nanostructured Heparin Conjugated-Polycaprolactone (HPCL) on the differentiation of Adipose Mesenchymal Stem Cells (ADMSCs) into Smooth Muscle Cells (SMCs). Methods: PCL nanofibers were produced by electrospinning and conjugated with heparin. The surface properties of the scaffolds were analyzed using FE-SEM for imaging, FTIR for investigation of chemical structure, assessments of surface roughness, thermomechanical characteristics, and the contact angle to evaluate wettability. The study included six groups; Group I: ADMSCs cells, Group II: ADMSCs seeded on PCL, Group III: ADMSCs seeded on HPCL, Group IV: SMCs cells, Group V: SMCs seeded on PCL, and Group VI: MSCs seeded on HPCL. SMCs differentiation was evaluated using specific genes and proteins. Results: Immobilization of heparin was confirmed by amide I (3427 cm􀀀 1) and it was significantly raised on HPCL scaffolds. SMCs + HPCL group revealed the highest cell viability and the most significant increase in differentiation-related genes and proteins at 7 days (p < 0.05). Conclusion: HPCL nanofibers have an effect on SMCs differentiation, which are the functional units of the ureter. Therefore, they could represent a valuable tool for tissue engineering applications in ureter repair.