The functionality and structural integrity of the cardiovascular system profoundly depend on vascular smooth muscle cells (VSMCs). Human mesenchymal stem cells (hMSCs) have the potential to differentiate into VSMCs, which is promising for regenerative medicine and the production of vascular grafts. This study investigates the combined effect of micropatterned substrates and TGF-β1 on hMSC differentiation into VSMCs. After culturing hMSCs on both micropatterned and flat substrates for six days with or without TGF-β1 treatment, cell morphology and the expression of specific smooth muscle markers were assessed by immunofluorescence staining, RT-qPCR, and Western blotting. It was established that the hMSCs on micropatterned substrates with TGF-β1 promoted higher protein levels of smooth muscle myosin heavy chain (MYH11) than control hMSCs on flat substrates without TGF-β1(p-value < 0.001). Additionally, samples of micropatterned substrates with TGF-β1 treatment showed significantly enhanced expression of the MYH11 gene. Notably, hMSCs treated with TGF-β1 and cultured on flat substrates demonstrated even higher Cadherin-11 mRNA expression compared to cells cultured on both micropatterned and flat substrates without TGF-β1 (p-value < 0.05).. In addition, it was found that KLF4 protein was expressed at significantly higher levels in hMSCs seeded on the flat substrate without TGF-β1 treatment compare to those cultured on micropatterned substrate with TGF-β1 treatment (p-value < 0.001). Based on these observations, the study concludes that micropatterned substrates combined with TGF-β1 treatment preferentially promote the high expression of MYH11, representing an advanced state of smooth muscle cell organization. This combination also led to alterations in the level of KLF4 and up-regulation of cadherin-11 in the cells. This research offers valuable insights into MSC differentiation into VSMCs, potentially guiding the design of more effective vascular grafts that closely mimic native blood vessel properties. Significance: The incorporation of micropatterned substrates alongside TGF-β1 markedly improves the differentiation of hMSCs into VSMCs, revealing a synergistic interaction that refines cellular fate determinations. This investigation underscores the pivotal role of modulating KLF4 within the differentiation pathway, importance the importance of comprehension the fundamental molecular mechanisms to advance regenerative approaches. The integration of micropatterned substrates with TGF-β1 holds significant potential for enhancing tissue engineering endeavors focused on vascular regeneration, which may lead to better outcomes in the field of regenerative medicine.