Mesenchymal stem cells (MSCs) are a compatible cellular alternative for regenerative medicine and tissue engineering because of their powerful multipotency. Matrix stiffness plays a profound role on stem cell behavior. Nevertheless, the effect of matrix stiffness on umbilical cordmesenchymal stem cells (UC-MSCs) remains unexplored. To conduct an in-depth exploration, we cultured UC-MSCs on different stiffness (Young's modulus: 13-16, 35-38, 48-53, and 62-68 kPa) polyacrylamide gels coated with fibronectin. We found that the proliferation and adhesion of UC-MSCs varied when cultured on the different matrices, and the spreading capacity was stronger as the stiffness increased (*P < 0.05). Real-time quantitative PCR results showed that the soft matrix promoted adipogenic differentiation, with higher expression levels of adipocytic markers like PPAR. and C/EBPa (*P < 0.05). In contrast, cells tended to differentiate into muscle when cultured on the 48-53 kPa matrix, which was validated by increased expression of myogenic makers like desminand MOYG (*P < 0.05). Moreover, increased expression of osteoblastic makers (*P < 0.05), such as ALP, collagen type I, osteocalcin, and Runx2, confirmed that cells differentiated into bone on the high-stiffness matrix.