Accumulatively, cellular behaviours triggered by biochemical cues have been widely explored, and the focus of research is gradually shifting to biophysical cues. Compared to physical parameters such as stiffness, substrate morphology, and viscoelasticity, the influence of viscosity on cellular behaviours is relatively unexplored and overlooked. Thus, in this study, the influence of viscosity on the adipogenic and osteogenic differentiation of human mesenchymal stem cells (hMSCs) was investigated by adjusting the viscosity of the culture medium. Viscosity exhibited different effects on adipogenic and osteogenic differentiation of hMSCs during two-dimensional (2D) culture. High viscosity facilitated cell osteogenic while inhibiting adipogenic differentiation. During adipogenic differentiation, the effect of viscosity on cell proliferation was negligible. However, during osteogenic differentiation, high viscosity decreased cell proliferation. The different influence of viscosity could be explained by the activation of mechanotransduction regulators of Yes-associated protein (YAP) and β-catenin. High viscosity could promote YAP and β-catenin nuclear translocation, which was responsible for the increased osteogenesis and decreased adipogenesis. This study could broaden the understanding of how viscosity can affect stem cell differentiation during 2D culture, which is valuable for tissue engineering.